Transcription factor FOXP2 is a flow‐induced regulator of collecting lymphatic vessels
2021; Springer Nature; Volume: 40; Issue: 12 Linguagem: Inglês
10.15252/embj.2020107192
ISSN1460-2075
AutoresMagda Nohemí Hernández-Vásquez, Maria H. Ulvmar, Alejandra González‐Loyola, Ioannis Kritikos, Ying Sun, Liqun He, Cornelia Halin, Tatiana V. Petrova, Taija Mäkinen,
Tópico(s)Lymphatic System and Diseases
ResumoArticle2 May 2021Open Access Source DataTransparent process Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels Magda N Hernández Vásquez Magda N Hernández Vásquez Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Maria H Ulvmar Maria H Ulvmar Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Alejandra González-Loyola Alejandra González-Loyola Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland Search for more papers by this author Ioannis Kritikos Ioannis Kritikos Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland Search for more papers by this author Ying Sun Ying Sun Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Liqun He Liqun He Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Cornelia Halin Cornelia Halin Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland Search for more papers by this author Tatiana V Petrova Tatiana V Petrova Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland Search for more papers by this author Taija Mäkinen Corresponding Author Taija Mäkinen [email protected] orcid.org/0000-0002-9338-1257 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Magda N Hernández Vásquez Magda N Hernández Vásquez Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Maria H Ulvmar Maria H Ulvmar Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Alejandra González-Loyola Alejandra González-Loyola Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland Search for more papers by this author Ioannis Kritikos Ioannis Kritikos Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland Search for more papers by this author Ying Sun Ying Sun Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Liqun He Liqun He Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Cornelia Halin Cornelia Halin Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland Search for more papers by this author Tatiana V Petrova Tatiana V Petrova Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland Search for more papers by this author Taija Mäkinen Corresponding Author Taija Mäkinen [email protected] orcid.org/0000-0002-9338-1257 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Author Information Magda N Hernández Vásquez1, Maria H Ulvmar1, Alejandra González-Loyola2, Ioannis Kritikos3, Ying Sun1, Liqun He1, Cornelia Halin3, Tatiana V Petrova2 and Taija Mäkinen *,1 1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden 2Vascular and Tumor Biology Laboratory, Department of Oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne, Switzerland 3Institute of Pharmaceutical Sciences, ETH Zürich, Zürich, Switzerland *Corresponding author. Tel: +46 18 471 41 51; E-mail: [email protected] The EMBO Journal (2021)40:e107192https://doi.org/10.15252/embj.2020107192 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow-responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial-specific Tie2-Cre or the tamoxifen-inducible LEC-specific Prox1-CreERT2 line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow-induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel-type-specific endothelial cell identities. SYNOPSIS Molecular mechanisms that regulate the identity of functionally specialized lymphatic vessel types are incompletely understood. The current study identifies transcription factor FOXP2 as a signature gene and a critical transcriptional regulator of collecting lymphatic vessels. Transcriptome analysis of murine endothelial cells identifies specific expression of Foxp2 in collecting lymphatic vessels. FOXP2 expression is induced after initiation of lymph flow and regulated by fluid shear stress. Genetic deletion of Foxp2 in mice leads to collecting lymphatic vessel and valve defects. FOXP2 acts downstream of the flow-responsive transcription factor FOXC2 to regulate NFATc1 activity. Introduction The lymphatic vascular system maintains tissue fluid balance and immune homeostasis through a coordinated action of lymphatic capillaries (also known as initial lymphatics) and collecting lymphatic vessels. The blind-ended lymphatic capillaries are composed of endothelial cells with discontinuous button junctions that allow entry of excess extracellular fluid and immune cells (reviewed in (Potente & Mäkinen, 2017; Oliver et al, 2020; Petrova & Koh, 2020)). In contrast, the lymphatic capillary-draining collecting vessels have continuous zipper junctions that prevent excess leakage of fluid. Additional unique characteristics of the collecting lymphatic vessels are the smooth muscle coverage that facilitates fluid propulsion through contractions and the existence of bicuspid valves that prevent fluid backflow (Zawieja, 2009). The establishment of a hierarchy of functionally specialized vessel types is critical for normal lymphatic vascular function and, consequently, failure in this process can lead to lymphatic diseases. During embryonic development, lymphatic vessel formation occurs through transdifferentiation of venous into lymphatic endothelial cells (LECs), with contribution of additional non-venous sources in certain organs (reviewed in (Ulvmar & Mäkinen, 2016; Kazenwadel & Harvey, 2018)). The key regulator of LEC fate is the homeobox transcription factor PROX1 that is required for the formation of all lymphatic vascular beds (Wigle & Oliver, 1999). PROX1 co-operates with other transcription factors in regulating the LEC lineage-specific transcriptome. For example, the venous endothelial cell (EC) fate regulator COUP-TFII (You et al, 2005) is required for initiation of PROX1 expression and regulates lymphatic vessel formation through heterodimerization with PROX1 (Lee et al, 2009; Yamazaki et al, 2009; Lin et al, 2010; Srinivasan et al, 2010). The subsequent maturation of the primitive lymphatic vascular plexus into functional collecting vessels is controlled by an interplay between mechanical forces, induced by initiation of lymph flow, and another set of transcription factors including GATA2 and FOXC2 (Sabine et al, 2012; Kazenwadel et al, 2015). Oscillatory shear stress, mimicking turbulent flow in the branched network of primitive vessel plexus, induces upregulation of GATA2 and FOXC2 (Sabine et al, 2012; Kazenwadel et al, 2015). These factors subsequently regulate a transcriptional program required for the formation of lymphatic valves. Deficiency of GATA2 or FOXC2, or mutations in their DNA-binding domains consequently cause abnormal development of lymphatic valves and underlie human hereditary lymphedemas (Petrova et al, 2004; Ostergaard et al, 2011; Kazenwadel et al, 2012, 2015). While the mechanisms that specify and sustain the identity of the phenotypically distinct valve LECs are characterized with increasing detail (Geng et al, 2017), the pathways that specify the functionally different collecting vessels and lymphatic capillaries have not been delineated. Here, we aimed to determine the mechanism that regulates the functional specification of collecting lymphatic vessels through transcriptional analysis of LECs of distinct vessel subtypes. We identified forkhead box protein P2 (FOXP2), previously implicated in the development of speech and language in humans (Co et al, 2020), as a collecting vessel-specific transcription factor. Using cultured primary LECs and genetic mouse models, we further show that FOXP2 is transcriptionally induced by flow-mediated shear stress and regulates collecting lymphatic vessel and valve morphogenesis through co-operation with the major flow-responsive FOXC2/NFATc1 signaling pathway. Our results highlight the existence of unique transcription factor codes in the establishment of vessel-type-specific LEC identities that may provide an opportunity to exploit for therapeutic restoration of specific vessel functions. Results Global transcriptome analysis of endothelial cells of lymphatic vessel subtypes To identify genes regulating the functional specification of collecting lymphatic vessels, we performed transcriptome profiling of dermal ECs isolated from adult mouse ear skin by flow cytometry. Dermal cell suspensions from mice carrying a LEC reporter Prox1-GFP were first subjected to enrichment for ECs using PECAM1 antibody-coated magnetic beads, followed by sorting of live blood EC (BEC) and LEC populations based on the expression of the lymphatic markers PDPN, LYVE1, and Prox1-GFP (Fig 1A). LEC subpopulations were further defined by the level of LYVE1 expression (Mäkinen et al, 2005) and assigned as LYVE1high lymphatic capillaries, LYVE1low/− collecting vessels, and LYVE1intermed pre-collecting vessels (Fig 1A). Capillary LECs accounted for a majority (60.9 ± 0.2% [n = 3]) of the LEC population (Fig EV1A). Figure 1. Transcriptome analysis of lymphatic vessels identifies FOXP2 as a collecting vessel-specific transcription factor Isolation of dermal EC subtypes from the ear skin of 5-week-old Prox1-GFP mice using multicolor flow cytometry. Whole-mount immunofluorescence for LYVE1 (on the left) defines three Prox1-GFP+ lymphatic vessels subtypes that are sorted using the indicated gating scheme (on the right): LYVE1low/− collecting vessel (green arrow, col); LYVE1intermed pre-collecting vessel (gray arrow, pre-col); LYVE1high lymphatic capillary (magenta arrow, cap). Top 100 genes upregulated in collecting in comparison to lymphatic capillary LECs. Expression in BECs is shown for comparison. Heat map color coding shows log2 fold change. Red box indicates LEC-specific/enriched collecting vessel signature genes that include Foxp2 (red arrow). Whole-mount immunofluorescence of ear skin, mesentery, and flank, showing nuclear FOXP2 staining in collecting lymphatic vessels (arrows, col), but not in LYVE1+ lymphatic capillaries (cap) or blood vessels (bv). qRT–PCR analysis of Foxp1–4 in murine ECs freshly isolated from P11 mesentery (n = 6 mice, individual data points shown). Data are presented as mean relative expression (normalized to Gapdh) ± SD. Transcript levels for each transcript are presented relative to levels in LECs. P, Student's t-test. nd, not detected. Data information: Scale bar: 75 µm (A), 50 µm (C). Source data are available online for this figure. Source Data for Figure 1 [embj2020107192-sup-0007-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Transcriptome analysis of dermal endothelial cells Proportion of LEC subpopulations (of the total LEC population) sorted from mouse ear skin based on expression of LYVE1, PDPN, and Prox1-GFP as shown in Fig 1A (n = 3 samples). Horizontal line indicates mean. Col, collecting vessel; pre-col, pre-collecting vessel; cap, lymphatic capillary. Heat map showing differential expression of 1,191 genes between dermal LEC and BEC. Color coding shows log2 fold change. Gene ID list is provided in Dataset EV1. Intensity bar plots of cell lineage/phenotype genes showing expression in dermal EC populations (each bar represents one sample, n = 3). Y-axis represents expression level (signal intensity value given in arbitrary units (AU)). Intensity bar plot of Foxp2 showing expression in dermal EC populations (n = 3). Y-axis represents expression level (signal intensity value given in arbitrary units (AU)). Download figure Download PowerPoint Affymetrix GeneChip analysis of the isolated EC populations revealed 1,191 genes that were differentially expressed (FDR < 0.02, log2 fold change > 1 or < −1) between BECs and (all) LECs, including the established BEC-LEC lineage markers Flt1, Nrp1, and Prox1 (Fig EV1B and C, Dataset EV1). As expected, Lyve1 was highly expressed in LECs of lymphatic capillaries and pre-collecting vessels (Fig EV1C). In contrast, the valve-LEC-specific Cldn11 (Takeda et al, 2019) was highly expressed in LECs of collecting and pre-collecting vessels (Fig EV1C). Pdgfrb expression suggested contamination of the BEC, but not LEC population with mural cells (Fig EV1C), which is commonly observed in bulk-sorted BECs due to close association of the two cell types (Vanlandewijck et al, 2018). No significant contamination by Lum+ fibroblasts was observed in any of the EC populations (Fig EV1C). These results indicate a successful generation of a microarray dataset for differential gene expression analysis of LECs of lymphatic capillaries and collecting vessels (Dataset EV2). The transcription factor FOXP2 is specifically expressed in endothelial cells of collecting lymphatic vessels Next, we focused on collecting lymphatic vessel enriched genes (Fig 1B, Dataset EV3) that also showed LEC-specific pattern of expression with no or low expression in the BECs (Fig 1B, boxed). Among these was the gene encoding the transcription factor forkhead box protein P2 (FOXP2; Figs 1B and EV1D) that has previously been studied in the nervous system and implicated in several cognitive functions including development of speech and language in humans ((Lai et al, 2001), reviewed in (Co et al, 2020)), and lung development (Shu et al, 2007). Its role in other organ systems, including the vasculature, is not known. Selective expression of Foxp2 in collecting lymphatic vessels suggested involvement in establishing vessel-type-specific LEC identity. To first validate the transcriptome data, we performed whole-mount immunofluorescence staining of adult mouse ear skin. Staining with antibodies against FOXP2 revealed nuclear expression in LYVE1− collecting lymphatic vessels, but not in LYVE1+ lymphatic capillaries or in blood vessels (Fig 1C). Uniform expression of FOXP2 was detected along the collecting vessel, including the luminal valves composed of LECs expressing high levels of PROX1 (Fig 1C and Appendix Fig S1A). FOXP2 was also expressed in the mesenteric collecting vessels and large flank collectors (Fig 1C), but not in lymphatic capillaries of the intestinal villi (lacteals) or the diaphragm (Appendix Fig S1B), or in the "lymphatic-like" Schlemm's canal in the eye (Appendix Fig S1C). qRT–PCR analysis of BECs and LECs freshly isolated from the mesentery of P11 mice by flow cytometry further demonstrated that Foxp2 was the only differentially expressed member of the FOXP family of transcription factors (Fig 1D), which was also supported by the dermal array data (Appendix Fig S1D). The closely related Foxp1 and Foxp4 were expressed in both BECs and LECs, while Foxp3 was not detected (Fig 1D). Taken together, these results identify FOXP2 as a potential new transcriptional regulator of collecting lymphatic vessel identity and morphogenesis. FOXP2 expression is regulated by flow To investigate whether FOXP2 is functionally important for collecting lymphatic vessel formation, we first analyzed its expression in the developing vasculature. We studied the mesenteric lymphatic vessels that begin to form at embryonic (E)13 through lymphvasculogenic assembly of LEC progenitors (Stanczuk et al, 2015), and undergo remodeling and valve morphogenesis after the onset of lymph flow at E15-E16 (Bazigou et al, 2009; Norrmén et al, 2009; Sabine et al, 2015). Whole-mount immunofluorescence staining of embryonic mesenteries showed no expression of FOXP2 between E15-E16 (Fig 2A), suggesting that FOXP2 is not involved in the early steps of collecting vessel formation. FOXP2 expression was first observed at E17, and its expression was maintained in the postnatal mesenteric lymphatic vessels (Figs 1C and 2A). FOXP2 expression was not restricted to PROX1high valves but showed a uniform pattern of expression in all LECs of collecting vessels from E17 onwards. A similar pattern of FOXP2 expression was observed in embryonic skin. FOXP2 was not expressed at E14 or E15, when a primary dermal lymphatic capillary plexus forms through vessel sprouting, but was upregulated at E18, when remodeling of lymphatic capillaries into collecting vessels is initiated (Fig EV2). Figure 2. FOXP2 expression is regulated by flow A. Whole-mount immunofluorescence of embryonic mesenteries of the indicated developmental stages. Note induction of FOXP2 expression at E17. B. Whole-mount immunofluorescence of E18 mesenteries fixed immediately after dissection (left panel) or after 24 h of ex vivo culture (right panel). Note loss of patterning of PROX1high valve LECs and downregulation of FOXP2 expression in flow-abrogated vessels after ex vivo culture. C, D. Immunofluorescence (C) and qRT–PCR analysis (D) of HDLECs grown under static conditions or exposed to OSS for 48 h (n = 3 independent experiments). Data are presented as mean ± SD. P, Student's t-test. Data information: Scale bar: 100 µm (A, B), 50 μm (C). Source data are available online for this figure. Source Data for Figure 2 [embj2020107192-sup-0008-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. FOXP2 expression in the developing dermal vasculature Whole-mount immunofluorescence of embryonic back skin at the indicated developmental stages. Note expression of FOXP2 (single channel images on the right) in the developing collecting vessel (arrow) including luminal valve (asterisk) at E18. Scale bar: 50 µm. Download figure Download PowerPoint To investigate if the induction of FOXP2 expression in vivo was related to initiation of flow in mesenteric lymphatic vessels at E15–E16, we first utilized ex vivo culture of E18 mesenteries as a model of abrogated flow (Sabine et al, 2012). As expected, ex vivo culture of vessels for 24 h led to loss of patterned valve regions composed of PROX1high LECs, although PROX1 expression level was not affected (Fig 2B). This coincided with the downregulation of FOXP2 (Fig 2B), suggesting that maintenance of FOXP2 levels/expression is dependent on flow. To directly test if flow regulates FOXP2 expression, we exposed primary human dermal LECs (HDLECs) to different types of fluid shear stress implicated in collecting lymphatic vessel and valve morphogenesis. Oscillatory shear stress (OSS) has previously been used to mimic disturbed flow in the developing lymphatic network and shown to regulate the transcriptional program controlling valve morphogenesis (Sabine et al, 2012, 2015). On the other hand, laminar shear stress (LSS) regulates lymphatic vessel remodeling as well as LEC proliferation and quiescence (Wang et al, 2016; Choi et al, 2017a, 2017b; Geng et al, 2020). Under static conditions, only a weak immunofluorescence signal for FOXP2 protein was detected in HDLECs (Fig 2C). However, exposure to OSS robustly induced FOXP2 protein (Fig 2C) and FOXP2 mRNA (Fig 2D). Notably, this was observed at 48 h but not earlier (Fig EV3A), whereas the OSS-regulated GATA2 (Fig EV3A) was upregulated already at 24 h, as previously reported (Sweet et al, 2015). LSS led to a more modest upregulation of FOXP2 (Fig EV3B). Increased expression of genes previously shown to be regulated by OSS (GJA4 (Sabine et al, 2012), Fig 2D) or LSS (KLF4 (Choi et al, 2017a), Fig EV3B) in LECs was also observed. Click here to expand this figure. Figure EV3. Regulation of FOXP2 expression by oscillatory and laminar flow A, B. qRT–PCR analysis of HDLECs grown under static conditions or exposed to oscillatory shear stress (OSS) for the indicated times (A), or to laminar shear stress (LSS) at the indicated dyn/cm2 for 48 h (n = 4 independent experiments). GATA2 and KLF4 are shown as controls. Data are presented as mean ± SD. P, Student's t-test. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Analysis of FOXP2 expression and function in venous and lymphovenous valves A, B. Whole-mount immunofluorescence of venous valves of the proximal femoral vein in P7 Foxp2flox/flox;Tie2-Cre (A) and Foxp2flox/−;Tie2-Cre (B) mice with respective littermate controls. Note expression of FOXP2 in ECs of the valve leaflet but apparently normal PROX1high integrinα9+ valve leaflets in Foxp2-deficient mice. C. Left: Immunofluorescence of a coronal vibratome section of an E14 Vegfr3-CreERT2;R26-mTmG (Martinez-Corral et al, 2016) embryo showing no expression of FOXP2 in LVV (arrows). FOXP2+ cells are likely (PROX1−) neuronal cells. Maximum intensity projection is shown above, and a single optical section at the level of LVV leaflets below. GFP expression in LECs was induced by intraperitoneal injection of 1 mg of 4-OHT into pregnant female. Right: Visualization of FOXP2+ neuronal cells confirmed successful staining (arrowheads). LS, lymph sac; IJV, internal jugular vein; SVC, superior vena cava; A, artery. Data information: Scale bar: 50 µm (A–C, lymphovenous connection), 200 µm (C, brain). Download figure Download PowerPoint Taken together, these results demonstrate that FOXP2 expression is induced in vivo after the onset of flow, and regulated in vitro by OSS and, to a lesser extent, laminar flow. Endothelial-specific deletion of Foxp2 leads to collecting lymphatic vessel defects In order to investigate the potential role of FOXP2 in collecting lymphatic vessel development, we genetically deleted it in all ECs using the Tie2-Cre mice in combination with the floxed Foxp2 allele (Fig 3A). The organization and gross morphology of the blood and lymphatic vessel networks in adult ear or embryonic back skin were not affected by loss of Foxp2 (Appendix Fig S2A–C). Analysis of early postnatal mesenteric vasculature also revealed grossly normal collecting lymphatic vessel morphology in the Foxp2flox/flox; Tie2-Cre+ mutant mice in comparison with littermate controls (Fig 3B). However, staining for lymphatic valve marker integrin-α9 (Bazigou et al, 2009) revealed shortened valve leaflets in the mutant mice (Fig 3B). Notably, FOXP2 expression was efficiently depleted in the collecting vessels of the mutant mice except for the valves. Unexpectedly, in four out of five mutant mice analyzed the majority of the valves (85 ± 30%, n = 4 mice, 19 vessels) were formed of FOXP2+ cells (Fig 3C). Valve-specific selection of non-recombined LECs suggests selective requirement of FOXP2 in their morphogenesis. Figure 3. Endothelial-specific deletion of Foxp2 leads to defective collecting vessel and valve morphogenesis A. Genetic constructs and breeding strategies used to generate pan-endothelial Foxp2flox/flox;Tie2-Cre (gray) or Foxp2flox/−;Tie2-Cre (blue) deletion. B, C. Whole-mount immunofluorescence of P7 mesenteries, showing abnormally short integrin-α9+ lymphatic valve leaflets (B) and selection of non-recombined FOXP2+ LECs (C) in Foxp2flox/flox;Tie2-Cre vessels. D. Whole-mount immunofluorescence of P7 mesenteries, showing efficient FOXP2 depletion in Foxp2flox/−;Tie2-Cre vessels. Dotted line indicates vessel width. E. Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox/−;Tie2-Cre and littermate controls (n = vessels [mice], as indicated). F. Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox/−;Tie2-Cre mice. A spectrum of defects in the mutants ranging from shortened integrin-α9+FN-EIIIA+ valve leaflets (middle panels) to a complete loss of leaflets (right panels) are shown. G, H. Quantification of the proportion of valves with integrin-α9+ leaflets (G), and leaflet length (H) in P7 Foxp2flox/−;Tie2-Cre and littermate controls (n = valves [mice], as indicated). Leaflet length = 0 corresponds to valves with no leaflets. I. Images of P7 intestine (on the left) and mesentery (on the right) showing submucosal reflux (arrows) and leakage (arrowheads) in the Foxp2flox/−;Tie2-Cre mice. J. Chyle phenotypes in P7 Foxp2flox/−;Tie2-Cre and littermate controls (n = number of mice as indicated). Data information: In (E, G, H), data are presented as mean ± SD. P, Student's t-test. In (J), P, Fisher's exact test. Scale bar: 100 µm (B, D), 50 µm (C, F), 500 µm (I). Source data are available online for this figure. Source Data for Figure 3 [embj2020107192-sup-0009-SDataFig3.xlsx] Download figure Download PowerPoint To maximize gene targeting efficiency, we generated mice carrying the floxed in combination with a null Foxp2 allele (Fig 3A). Efficient FOXP2 depletion in LECs, including the valves, in the Foxp2flox/−; Tie2-Cre+ mice resulted in a spectrum of valve defects as well as increased collecting lymphatic vessel width (Fig 3D and E). A proportion (~ 25%) of valves composed of PROX1high LECs had no leaflets, as assessed by staining for integrin-α9 and its ligand fibronectin-EIIIA (Fig 3F and G). Quantification showed reduced leaflet length in the remaining valves in the mutants compared to littermate controls at this stage (Fig 3F and H). The mutant mice also showed chyle accumulation in the submucosal lymphatic vessel network on the intestinal wall, suggesting backflow from the mesenteric collecting lymphatic vessels, as well as chyle leakage from collecting vessels (Fig 3I and J). These defects did not, however, compromise postnatal survival and growth in the mutant mice, and analysis of adult mesenteries revealed morphologically normal valves in the Foxp2flox/−; Tie2-Cre+ mice (Appendix Fig S2D). In agreement with the lack of expression of FOXP2 in lacteal LECs (Appendix Fig S1B), we could not observe defects in these vessels in the Foxp2flox/−; Tie2-Cre+ mice (Appendix Fig S2E). Together, these data demonstrate a critical requirement of Foxp2 for collecting lymphatic vessel and valve morphogenesis. Loss of FOXP2 leads to reduced expression of known regulators of lymphatic and valve development FOXP2 is required in the nervous system for several cognitive functions, including general brain development and synaptic plasticity (Kim et al, 2019; Co et al, 2020). Interestingly, validated direct FOXP2 targets in the nervous system include genes encoding known regulators of lymphatic development (NRP2) and valve morphogenesis (EphrinB2 and SEMA3A) (Mäkinen et al, 2005; Xu et al, 2010; Vernes et al, 2011; Bouvrée et al, 2012; Jurisic et al, 2012; Ochsenbein et al, 2014). Immunofluorescence staining of P6 mesenteric collecting vessels showed marked downregulation of NRP2 in Foxp2flox/−; Tie2-Cre+ mice compared to littermate controls (Figs 4A and B). In addition, RNAscope-based whole-mount in situ hybridization revealed a significant reduction of Efnb2 transcript in Foxp2-deficient lymphatic vessels (Fig 4C and D). FOXP2 thus regulates genes critical for normal lymphatic vessel and valve development. Figure 4. Downregulation of FOXP2 targets Nrp2 and Efnb2 in Foxp2-deficient lymphatic vessels A, B. Whole-mount immunofluorescence (A) and quantification of NRP2 staining intensity (B) in P6 mesenteric lymphatic vessels of Foxp2flox/−;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated). C, D. RNAscope-based whole-mount in situ hybridization (C) and quantification of Efnb2 transcript levels (D) in P7 mesenteric lymphatic vessels of Foxp2flox/−;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated). Data information: In (B, D), data are presented as mean ± SD. P, Student's t-test. Scale bar: 100 µm. Source data are available online for this figure. Source Data for Figure 4 [embj2020107192-sup-0010-SDataFig4.xlsx] Download figure Download PowerPoint FOXP2 is required LEC-autonomously for valve formation and maintenance To study the potential fun
Referência(s)