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• W4367018414 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Multiple factors are required to form functional lymphatic vessels. Here, we uncover an essential role for the secreted protein Svep1 and the transmembrane receptor Tie1 during the development of subpopulations of the zebrafish facial lymphatic network. This specific aspect of the facial network forms independently of Vascular endothelial growth factor C (Vegfc) signalling, which otherwise is the most prominent signalling axis in all other lymphatic beds. Additionally, we find that multiple specific and newly uncovered phenotypic hallmarks of svep1 mutants are also present in tie1, but not in tie2 or vegfc mutants. These phenotypes are observed in the lymphatic vasculature of both head and trunk, as well as in the development of the dorsal longitudinal anastomotic vessel under reduced flow conditions. Therefore, our study demonstrates an important function for Tie1 signalling during lymphangiogenesis as well as blood vessel development in zebrafish. Furthermore, we show genetic interaction between svep1 and tie1 in vivo, during early steps of lymphangiogenesis, and demonstrate that zebrafish as well as human Svep1/SVEP1 protein bind to the respective Tie1/TIE1 receptors in vitro. Since compound heterozygous mutations for SVEP1 and TIE2 have recently been reported in human glaucoma patients, our data have clinical relevance in demonstrating a role for SVEP1 in TIE signalling in an in vivo setting. Editor's evaluation This study presents strong and compelling evidence that the extra-cellular matrix protein SVEP-1 interacts with the TIE1 receptor to promote aspects of lymphangiogenesis that are independent of canonical VEGF-C signaling. Using zebrafish models to show genetic interactions and cells to provide evidence of biochemical interaction, the study shows a functional requirement for these genes/proteins in specific aspects of lymphangiogenesis. These novel findings will be of interest to developmental and cell biologists and to those studying lymphatic disease as it potentially provides novel therapeutic targets. https://doi.org/10.7554/eLife.82969.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The lymphatic system is part of the vasculature and provides essential functions for tissue fluid homeostasis, absorption of dietary fats, and immune surveillance. Malfunction of the lymphatic vasculature can lead to severe lymphedema, obesity, or chronic inflammatory diseases (Mäkinen et al., 2021; Oliver et al., 2020). Since treatment options are rare and often only transiently effective, understanding the molecular mechanisms driving lymphangiogenesis is a prerequisite for the development of new therapeutic approaches (Mäkinen et al., 2021). To that end, mice and zebrafish have served as popular model organisms to study the development of lymphatic vessels and are commonly used for analyzing the underlying genetic and molecular mechanisms (Mäkinen et al., 2007; Padberg et al., 2017; van Impel and Schulte-Merker, 2014). Furthermore, many genes that are essential for lymphangiogenesis in zebrafish are evolutionarily conserved. Their inactivation leads to lymphatic defects in zebrafish and mice, and mutations in their orthologues are causative for human diseases (Alders et al., 2009; Gordon et al., 2013; Hogan et al., 2009; Mauri et al., 2018; Wang et al., 2020). In the trunk vasculature of the zebrafish, so-called lympho-venous sprouts arise from the posterior cardinal vein at 32 hours post-fertilization (hpf). They migrate dorsally and either remodel an intersegmental artery into a vein, or they migrate along the so-called horizontal myoseptum (HM) as parachordal lymphangioblasts (PLs) at 2 days post fertilization (dpf). At 3 dpf, PLs migrate dorsally and ventrally to form the trunk lymphatic vasculature, consisting of the dorsal longitudinal lymphatic vessel, the intersegmental lymphatic vessels, and the thoracic duct (TD) (Hogan et al., 2009; Hogan and Schulte-Merker, 2017; Padberg et al., 2017). A separate lymphatic network, the facial lymphatics, arises in a distinctly different manner, originating from three progenitor populations: (1) the primary head sinus-lymphatic progenitors (PHS-LP), (2) a migratory angioblast cell near the ventral aorta, and (3) the major population sprouting from the common cardinal vein (CCV) (Eng et al., 2019). These progenitor populations proliferate, migrate and connect to each other in a relay-like mechanism (Eng et al., 2019). A third lymphatic bed is composed of the brain lymphatic endothelial cells (BLECs), which are single endothelial cells residing within the leptomeningeal layer of the zebrafish brain and that arise from the choroidal vascular plexus (Bower et al., 2017; van Lessen et al., 2017; Galanternik et al., 2017). During larval stages, BLECs are often positioned next to meningeal blood vessels and stay at the distal periphery of the optic tectum and other brain regions (van Lessen et al., 2017). However, molecular mechanisms supporting the development of BLECs and facial lymphatics still need to be examined in more detail. The best-studied pathway driving lymphangiogenesis comprises the growth factor Vascular Endothelial Growth Factor C (VEGFC), which is secreted as a pro-form that is processed through the concerted activity of Collagen and Calcium-Binding EGF domain-containing protein 1 (CCBE1) (Bos et al., 2011; Hogan et al., 2009; Jeltsch et al., 2014; Le Guen et al., 2014; Roukens et al., 2015) and a disintegrin and metalloproteinase with thrombospondin motifs (Adamts) 3/14 (Jeltsch et al., 2014; Wang et al., 2020) in the extracellular space. Fully processed VEGFC binds to its receptor VEGFR3 as well as VEGFR2 and induces lymphangiogenesis (Joukov et al., 1997; Karkkainen et al., 2004). Apart from the VEGFC/VEGFR3 pathway, TIE-ANG signalling was shown to be essential for lymphangiogenesis and vessel remodelling in mice and humans. This signalling cascade is composed of two receptor tyrosine kinases, tyrosine-protein kinase receptor 1 (TIE1) (Partanen et al., 1992) and tyrosine endothelial kinase (TEK), also known as tyrosine-protein kinase receptor 2 (TIE2) (Dumont et al., 1993), and multiple angiopoietin ligands including angiopoietin 1 (ANG 1) (Davis et al., 1996; Suri et al., 1996) and angiopoietin 2 (ANG 2) (Maisonpierre et al., 1997). In mammals, TIE signalling is activated through binding of Angiopoietins to TIE2 (Davis et al., 1996; Maisonpierre et al., 1997). TIE1 can either block or activate the signalling cascade in a context-dependent manner by forming heterodimers with TIE2 (Hansen et al., 2010; Marron et al., 2000; Saharinen et al., 2005; Savant et al., 2015; Seegar et al., 2010). Tie1 knockout mice display haemorrhages from E13.5 to P0, which lead to death and are preceded by lymphatic defects and edema formation from E12.5 onwards (D’Amico et al., 2010). In contrast to Tie1 mutant mice, Tie2 mutant mice die already at E9.5–10.5 due to defective cardiac development and vascular remodelling (Dumont et al., 1994; Sato et al., 1995). Conditional knockout of Tie2 in lymphatic cells revealed the importance of TIE2 for lymphatic vessel development in mice especially for Schlemm’s canal formation (Kim et al., 2017; Thomson et al., 2014). Recently, Korhonen et al. showed that conditional Tie1 deletion, Tie1;Tie2 double deletion and Ang2 blocking resulted in impaired postnatal lymphatic capillary network development in mice (Korhonen et al., 2022). In zebrafish, tie2 mutants do not have any overt vascular defects (Gjini et al., 2011; Jiang et al., 2020), while tie1 mutants show cardiac morphogenesis and vascular defects (Carlantoni et al., 2021). In 2017, a new key player in lymphangiogenesis was discovered through genetic screens in zebrafish: sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1 (svep1), also referred to as polydom (Karpanen et al., 2017; Morooka et al., 2017). Svep1 encodes a large extracellular matrix molecule, with a total of 3571 amino acids and a variety of protein domains. The C terminal half of Svep1 mainly consists of complement control protein (CCP), also called sushi domain, repeats and EGF domains, indicating a possible role in protein-binding stabilization. Svep1−/− mice show normal development of the primitive lymphatic plexus until E12.5, but then fail to undergo remodelling of lymphatic vessels and formation of lymphatic valves at later embryonic stages, accompanied by edema formation and death postnatally (Karpanen et al., 2017; Morooka et al., 2017). Recently, Michelini et al. reported possible implications of SVEP1 in lymphedema formation in human patients, underlining the importance of SVEP1 for the lymphatic vasculature (Michelini et al., 2021). Additionally, SVEP1 is also required for Schlemm’s canal formation in mice (Thomson et al., 2021). In zebrafish, svep1 mutants exhibit a near-complete loss of the TD, demonstrating an essential function during lymphangiogenesis in zebrafish (Karpanen et al., 2017; Morooka et al., 2017). In the present study, we show defects in the lymphatic head vasculature in svep1 mutants, comprising a variable loss of BLECs and a specific facial lymphatic phenotype, which is complementary to the phenotypes observed in mutants of Vegfc/Vegfr3 pathway members. Therefore, we identified a lymphatic structure in the zebrafish that, in contrast to all other lymphatic structures, forms independently of the Vegfc/Vegfr3 pathway, but depends on Svep1. Murine SVEP1 has been shown to bind to the α9 form of integrin (ITGA9) as well as the TIE2 ligands ANG1 and ANG2 in vitro (Morooka et al., 2017; Sato-Nishiuchi et al., 2012). However, until now, putative interaction partners of Svep1 have not been confirmed in vivo. In the present study, we first characterized novel lymphatic and blood vasculature defects of tie1 mutants, and subsequently realized that all phenotypic traits are shared between tie1 and svep1 mutants. These observations raised the question whether Svep1 and Tie1 interact, a notion that we tested both genetically and on a protein biochemistry level. Our results provide the first in vivo evidence for svep1 and tie1 genetic interaction, thus placing Svep1 as an important regulator of Tie1 function. Additionally, we show the interaction of SVEP1 and TIE1 in vitro for the respective versions of the zebrafish and human proteins. Since recent clinical data suggested SVEP1 as a genetic modifier of TIE2-related primary congenital glaucoma (PCG) (Young et al., 2020), our results have clinical relevance and will further help to understand the molecular basis of PCG. Results Svep1 is required for facial collecting lymphatic vessel formation in a Vegfc-independent manner Since svep1 mutants had previously been analyzed for lymphatic defects only in the trunk vasculature, we examined the head vasculature of svep1 mutants to detect further possible malformations of the lymphatic system. At 5 dpf we observed that svep1 mutants showed specific facial lymphatic defects, which seemed to be complementary to the facial lymphatic defects found in mutants of the Vegfc/Vegfr3 pathway members (Figure 1A). While mutants for Vegfc/Vegfr3 pathway members like ccbe1, adamts3/14, and vegfc retained the facial collecting lymphatic vessel (FCLV) (red dotted line in Figure 1A, B) but lacked all other structures of the facial lymphatics, svep1 mutants showed a specific loss of the FCLV. All other parts of the mature facial lymphatic network (including lymphatic branchial arches, lateral facial lymphatic, medial facial lymphatic, and otolithic lymphatic vessel (blue dotted line in Figure 1A)) were only partially reduced in svep1 mutants. Although the formation of the FCLV was strongly affected in all svep1 mutants analyzed, the severity of the defects of facial lymphatic structures varied between individual svep1 mutant embryos (Figure 1—figure supplement 1). Only simultaneous interference of both the Vegfc and Svep1 signalling pathways completely blocked the development of all facial lymphatic structures (Figure 1—figure supplement 2). To further characterize the differential roles of Svep1 and Vegfc during the formation of the facial lymphatic network, we examined the expression patterns of svep1 and vegfc during sprouting of the PHS-LP, the progenitor cells of the FCLV, at 50 hpf using transgenic reporter lines. We detected svep1 expression in cells juxtaposed to the sprouting LECs around the PHS, which later will form the FCLV, while vegfc expression was more restricted to the lateral facial lymphatic sprout arising from the CCV in all embryos analyzed (Figure 1C, Figure 1—figure supplement 3). Taken together, these observations indicate a Vegfc-independent role of Svep1 during the development of distinct aspects of the facial lymphatics. Figure 1 with 3 supplements see all Download asset Open asset Svep1 is required for the development of the FCLV, in a Vegfc-independent manner. (A) Schematic representation of facial lymphatic network at 5 dpf and maximum intensity projection of confocal images of flt4:mCitrine positive svep1 mutants (n = 10) and siblings (n = 6), highlighting facial lymphatic structures at 5 dpf. Scale bar = 100 µm. Note the absence of the FCLV (red dotted line) in svep1 mutants whereas other facial lymphatic structures are less strongly affected (OLV, LFL, MFL, and LAA marked by blue dotted lines). (B) Confocal images of flt4:mCitrine positive facial lymphatics in vegfc (n = 19), ccbe1 (n = 5), and adamts3;adamts14 (n = 2) mutants at 5 dpf. Scale bar = 100 µm. (C) Confocal images of svep1 and vegfc expression domains during sprouting from the PHS at 2 dpf, with schematic representation of different lymphatic progenitor populations. svep1 is expressed in close proximity to sprouting PHS-LPs, while vegfc expressing cells are more concentrated on the LECs arising from the CCV. Arrows point to sprouting PHS-LP. Scale bar = 50 µm. Expression patterns were confirmed in six embryos each (Figure 1—figure supplement 3). CCV, common cardinal vein; dpf, days post-fertilization; FCLV, facial collecting lymphatic vessel; FLS, facial lymphatic sprout; hpf, hours post-fertilization; LAA, lymphatic branchial arches; LEC, lymphatic endothelial cell; LFL, lateral facial lymphatic; MFL, medial facial lymphatic; OLV, otolithic lymphatic vessel; PHS, primary head sinus; PHS-LP, primary head sinus lymphatic progenitor; VA, ventral aorta; VA-A, ventral aorta angioblast; VA-L, ventral aorta lymphangioblast; WT, wildtype. Svep1 is essential for sprouting of BLECs and is expressed in close proximity to BLECs Since Svep1 is required for the formation of facial lymphatic structures (Figure 1), we wondered whether it is also involved in the development of an additional set of lymphatic endothelial cells, the BLECs. In mutants of the Vegfc/Vegfr3 pathway, BLECs are completely absent (Bower et al., 2017; van Lessen et al., 2017). In svep1 mutants, BLECs were found to be absent in most cases, but some embryos showed either reduced numbers or – in rare cases – even wildtype-like numbers of BLECs at 3 dpf (Figure 2A, B). In line with the idea that svep1 is required for the sprouting and migration of BLECs, we observed svep1 expressing cells in close proximity to the migrating BLECs at 3 dpf (Figure 2C, D). Thus, there is close juxtaposition of svep1 expressing cells with migrating LECs in all developing lymphatic structures examined, including the PLs in the trunk (Karpanen et al., 2017). Figure 2 Download asset Open asset Svep1 is required for the sprouting of BLECs. (A) Confocal images of sprouting BLECs, marked by flt4:mCitrine, at 3 dpf in svep1 mutants and siblings. Asterisks mark missing BLECs in svep1 mutants. Scale bar = 100 µm. (B) Quantification of BLECs at 3 dpf on each side of the embryo showed that svep1 mutants have significantly less BLECs on one or both sides of the brain hemispheres compared to siblings. For statistical analysis, no BLECs were counted as 0, BLECs being present on only one hemisphere as 1, whereas BLECs being detectable on both brain hemispheres were included as 2, for each embryo (svep1+/+: n = 10; svep1+/−: n = 12; svep1−/−: n = 12). Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD), ****p < 0.0001, ns = not significant. Scale bar = 100 µm. (C) Confocal images of svep1:Gal4; UAS:RFP, showing svep1 expression immediately adjacent to BLECs, marked by arrowheads, at 3 dpf. Scale bar = 100 µm. (D) Magnification and reduced stack numbers of boxed area in (C). Arrowhead marks BLEC. Scale bar = 50 µm. BLEC, brain lymphatic endothelial cell; dpf, days post-fertilization; MsV, mesencephalic vein; PHS, primary head sinus;. svep1 and tie1 mutants show near-identical lymphatic defects Murine SVEP1 has been shown to bind the TIE2 ligands ANG1 and ANG2 in vitro and to regulate expression of Tie1 as well as Tie2 (Morooka et al., 2017). It also has been suggested to play a role in TIE2-related PCG (Young et al., 2020). Hence, we wanted to investigate the role of Tie signalling in zebrafish lymphangiogenesis in order to assess potential interactions with svep1 in an in vivo situation. Lymphatic defects have not been previously reported in zebrafish mutants for either tie1 or tie2 (Carlantoni et al., 2021; Gjini et al., 2011; Jiang et al., 2020). Given the fact that there seems to be a very specific requirement for svep1 in FCLV development, we analysed facial lymphatic structures of tie1 and tie2 mutants in direct comparison to svep1 mutants. Since tie1 mutants developed strong edema at 4 dpf (data not shown), we focused our analysis on lymphatic phenotypes at 2 and 3 dpf to exclude secondary effects on the lymphatic vasculature. Significantly, tie1 mutant embryos showed the same facial lymphatic defects as svep1 mutant embryos at 3 dpf (Figure 3A), with the FCLV being strongly affected. We confirmed this observation also in a lyve1:DsRed transgenic background (Figure 3—figure supplement 1). This finding suggests that Tie1, either independently or in concert with Svep1 is responsible for FCLV formation in a Vegfc-independent manner. Examining other lymphatic cells, we found that tie1 mutants did not show any BLECs at 3 dpf and exhibited significantly reduced numbers of PLs at 2 dpf, similar to svep1 mutants (Figure 3B–E). Importantly, tie2 mutant embryos, when examined for the same anatomical features, were found to display normal facial lymphatics, BLECs and PL numbers (Figure 3A–C and E). Taken together, these findings demonstrate that loss of tie1, but not tie2, results in lymphatic defects highly similar to the ones seen in svep1 mutants, indicating that Svep1 constitutes an essential component acting in the Tie1 pathway. Figure 3 with 1 supplement see all Download asset Open asset tie1 mutants phenocopy the loss of svep1, while tie2 is dispensable for lymphangiogenesis. (A) Facial lymphatics at 3 dpf in flt4:mCitrine positive tie1, svep1 and tie2 mutants and sibling embryos (lateral view). Arrowheads point to FCLV and asterisks indicate the absence of FCLV. Scale bar = 100 µm. (B) flt4:mCitrine; flt1:tdTomato positive dorsal head vasculature in tie1, svep1, and tie2 mutants and in siblings at 3 dpf (dorsal view). In svep1 and tie1 mutants (but not in tie2 mutants) the presence of BLECs is strongly reduced. Arrowheads point to BLECs and asterisks indicate areas lacking BLECs. Scale bar = 100 µm. (C) Confocal images of PL cells, indicated by arrowheads, at 2 dpf in flt4:mCitrine; flt1:tdTomato positive tie1, svep1, and tie2 mutants and siblings, showing reduced PL numbers in svep1 and tie1 mutants. Asterisks indicate missing PLs. Scale bar = 100 µm. (D) Quantification of the presence of BLECs in tie1 mutants compared to siblings. (tie1+/+: n = 6; tie1+/−: n = 16; tie1−/−: n = 10) Mann–Whitney test was applied for statistical analysis. ***p = 0.001, ns = not significant. (E) Quantification of PL cell numbers in tie1 (tie1+/+: n = 9; tie1+/−: n = 23; tie1−/−: n = 14), svep1 (svep1+/+: n = 16; svep1+/−: n = 31; svep1−/−: n = 19), and tie2 (tie2+/+: n = 17; tie2+/−: n = 27; tie2−/−: n = 16) mutants compared to siblings. Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD), ****p < 0.0001, ns = not significant; BLEC, brain lymphatic endothelial cell; dpf, days post-fertilization; FCLV, facial collecting lymphatic vessel; PL, parachordal lymphangioblast. tie1 and svep1 mutants display identical PL cell migration and survival defects PLs first migrate along the HM and then start to migrate dorsally and ventrally along arteries to form the DLLV or the TD, respectively. Previously, it was shown that PLs in svep1 mutants fail to migrate dorsally or ventrally and rather remain at the HM (Karpanen et al., 2017). Here, we compared PL migration in svep1 and tie1 mutants using overnight imaging from 2.5 to 3.5 dpf to analyse if PLs in tie1 mutants phenocopy the PL migration defects of svep1 mutants (Figure 4A–L, Figure 4—videos 1–3). While around 40–50% of PLs in sibling embryos migrated along the artery, only 11% of PLs in tie1 and svep1 mutants showed migration in either dorsal or ventral direction along the artery (Figure 4M, N). Additionally, we observed around 33% apoptotic PLs in tie1 mutants and 55% in svep1 mutants. These apoptotic events could be a consequence of failed migration, or could be due to decreased survival as a direct consequence of absent Svep1 or Tie1 activity. To further characterize migration of PLs in svep1 and tie1 mutants, we tracked and plotted the migration route of individual PLs (Figure 4O, Q, Figure 4—figure supplements 1 and 2) and quantified the migration distance in the Y direction (i.e. migration in dorsal or ventral direction), mean velocity and total migration distance in tie1 and svep1 mutants (Figure 4P, R). PLs in svep1 as well as in tie1 mutants showed significantly less migration in ventral and dorsal directions compared to siblings, while the mean velocity and total migration distance were unchanged. Therefore, we can conclude that Svep1 and Tie1 are required for PL migration along the arteries in dorsal or ventral direction. Since we could observe the same specific migratory defects in both svep1 and tie1 mutants, these results further support a possible cross-talk between both proteins. Figure 4 with 5 supplements see all Download asset Open asset PL cell migration along arteries is severely affected in svep1 and tie1 mutants. (A–L) Still frames from confocal time-lapse imaging of embryos in a flt4:mCitrine; flt1:tdTomato transgenic background. (A–D) PL migration (indicated by arrowheads) of sibling embryo along aISV from 2.5 to 3.5 dpf. (E–H) Failed PL migration (indicated by asterisk) of svep1 mutants and (I–L) tie1 mutants along artery from 2.5 to 3.5 dpf. (M, N) Classification of PL migration along arteries. Statistical analysis was performed using Mann–Whitney test comparing the % of PL migration along arteries in each sibling and mutant embryo (sibling: n = 96 PLs in 18 embryos; svep1−/−: n = 36 PLs in 15 embryos; siblings: n = 52 PLs in 14 embryos; tie1−/−: n = 28 PLs in 10 embryos); ****p < 0.0001, ***p = 0.0003. (O, Q) Representative cell tracking routes (tracks centred to origin) of single PL cells marked by different colours in siblings (n = 17 PLs in 4 embryos; n = 7 in 2 embryos), tie1−/− (n = 5 PLs in 2 embryos) and svep1−/− (n = 6 PLs in 3 embryos). (P, R) Quantification of dorsal and ventral PL migration (delta Y migration distance), mean velocity and total migration distance in svep1 and tie1 mutants compared to sibling embryos excluding apoptotic PLs quantified in (M, N) revealed decreased migration in dorsal and ventral direction in svep1 (*p = 0.0148) as well as tie1 mutants (**p = 0.0023). ns = not significant; aISV, arterial intersegmental vessel; dpf, days post fertilization; HM, horizontal myoseptum; PL, parachordal lymphangioblast. Scale bar = 100 µm (D, H, L = 25 µm). tie1 mutants show blood vascular defects under reduced flow conditions While svep1 mutants were initially identified on the basis of their lymphatic phenotype (Karpanen et al., 2017), Coxam et al. recently showed that svep1 mutant embryos display unique vascular defects under reduced flow conditions (Coxam et al., 2022). Treatment of embryos with 0.014% tricaine between 30 and 48 hpf leads to incomplete formation of the dorsal longitudinal anastomotic vessel (DLAV) with gaps and non-lumenized DLAV segments at 2 dpf in svep1 mutant embryos. This phenotype is accompanied by increased Vegfa/Vegfr signalling and increased number of Apelin positive tip cells (Coxam et al., 2022). To investigate if tie1 mutants mimic this very specific and unusual vascular defect, we treated embryos from tie1 heterozygous parents with 0.014% tricaine between 30 and 48 hpf, and subsequently imaged the intersegmental vessels in the trunk. Our analysis showed that tie1 mutants treated with tricaine exhibited significantly more gaps and fewer lumenized DLAV segments (Figure 5D) compared to both untreated tie1 mutants (Figure 5B) and treated siblings (Figure 5C, E, F), suggesting that Svep1 and Tie1 might interact not only in lymphangiogensis but also during blood vessel development. For tie2 and vegfc mutants we did not observe any defects in DLAV formation upon tricaine treatment, indicating that this phenotype is specific for loss of Svep1 and Tie1 (Figure 5—figure supplement 1). Additionally, upon tricaine treatment, and even in untreated conditions, apelin expressing ECs were increased in ISVs of tie1 mutants as already shown for svep1 morphants treated with tricaine in Coxam et al., 2022 (Figure 5G–J). Since we observed increased apelin expressing ECs in tie1 mutants already in untreated conditions, we investigated if svep1 morphants also show increased apelin expression even without tricaine treatment (Figure 5I, J). svep1 morphants already showed increased apelin expression in the ISVs in untreated conditions ( Figure 5—figure supplement 2). We confirmed our results using in situ hybridization ( Figure 5—figure supplement 3). These observations indicate that apelin expression is affected in tie1 mutants as well as svep1 morphants, and support the hypothesis of Tie1 and Svep1 acting in the same molecular pathway. Figure 5 with 3 supplements see all Download asset Open asset Reduced blood flow leads to vascular anastomosis defects in tie1 mutants, similar to the defects in svep1 mutants. (A, B) Confocal images of sibling and tie1 mutant embryos at 2 dpf in a flt4:mCitrine and flt1:tdTomato transgenic background. (B’) Magnification and reduced stack of boxed area in (B). (C, D) Confocal images of sibling and tie1 mutant embryos treated with 0.014% tricaine from 30 until 48 hpf. Asterisks indicate incompletely formed DLAV segments. (D’) Magnification and reduced stack numbers of boxed area in (D). (E) Quantification of gaps in the DLAV in sibling and tie1 mutants that were either untreated or treated with 0.014% tricaine revealed significant increase of gaps in the DLAV in tie1 mutants. (F) Quantification of lumenized trunk segments of the DLAV in siblings and tie1 mutants, either untreated or treated with 0.014% tricaine (siblings untreated: n = 16; tie1−/− untreated: n = 20; siblings treated with 0.014% tricaine: n = 20; tie1−/− treated with 0.014% tricaine: n = 22), revealed significant decrease of lumenized segment numbers in the DLAV in tie1 mutants. Mann–Whitney test was applied for statistical analysis. (G, H) apelin:eGFP and flt1:tdTomato expression in 48-hpf-old embryos after tricaine treatment from 30 to 48 hpf and (I, J) in untreated conditions. (K) Maximum intensity projection of an aISV at 48 hpf, highlighting the ventral and dorsal region used for further quantifications in (J) adapted from Figure 5J of Coxam et al., 2022. (L) Quantification of ISVs with apelin expression in dorsal and ventral parts of the ISVs. Dorsal part was counted from DLAV until midline region. Lateral region was counted from midline region onwards in ventral direction. tie1 mutants showed significant increase of apelin positive ECs compared to siblings in untreated (dorsal: ***p = 0.0001; ventral: **p = 0.0028) and treated with 0.014% tricaine conditions (dorsal: **p = 0.0033; ventral: ***p = 0.0002) (siblings untreated: n = 53; tie1−/− untreated: n = 21; siblings treated with 0.014% tricaine: n = 66; tie1−/− treated with 0.014% tricaine: n = 28). Mann–Whitney test was applied for statistical analysis. Values are presented as means ± standard deviation (SD). ****p < 0.0001. Scale bar = 100 µm. hpf, hours post-fertilization; ISV, intersegmental vessel; DLAV, dorsal longitudinal anastomotic vessel; dpf, days post-fertilization. tie2 loss of function does not exacerbate the tie1 mutant phenotype To investigate a possible contribution of Tie2 to lymphatic Tie signalling as well as possible compensatory mechanisms, we examined tie1; tie2 double mutants at 2 dpf (Figure 6A–G). While tie1 mutants showed a highly significant reduction in PL numbers (Figure 6D, G), we found that an additional loss of one or two functional copies of tie2 did not further affect PL numbers in tie1 mutant embryos (Figure 6E–G). Additionally, loss of one tie1 allele in tie2 mutants did not result in any defects (Figure 6C, G). To further exclude contributions of Tie2 at later stages of lymphatic development on TD formation, we quantified the segm" @default.
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• W4367018414 date "2022-11-07" @default.
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• W4367018414 title "Decision letter: Svep1 is a binding ligand of Tie1 and affects specific aspects of facial lymphatic development in a Vegfc-independent manner" @default.
• W4367018414 doi "https://doi.org/10.7554/elife.82969.sa1" @default.
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