Bone morphogenetic proteins (BMPs) were originally identified as the active components in bone extracts that can induce ectopic bone formation. In recent decades, their key role has broadly expanded beyond bone physiology and pathology. Nowadays, the BMP pathway is considered an important player in vascular signaling. Indeed, mutations in genes encoding different components of the BMP pathway cause various severe vascular diseases. Their signaling contributes to the morphological, functional and molecular heterogeneity among endothelial cells in different vessel types such as arteries, veins, lymphatic vessels and capillaries within different organs. The BMP pathway is a remarkably fine-tuned pathway. As a result, its signaling output in the vessel wall critically depends on the cellular context, which includes flow hemodynamics, interplay with other vascular signaling cascades and the interaction of endothelial cells with peri-endothelial cells and the surrounding matrix. In this review, the emerging role of BMP signaling in lymphatic vessel biology will be highlighted within the framework of BMP signaling in the circulatory vasculature. Dysfunction of endothelial cells lining the inner wall of the circulatory and lymphatic vasculature is a major cause and amplifier of vascular disease. Mutations in genes encoding different components of the bone morphogenetic protein (BMP) pathway cause rare but severe vascular diseases. Most of these diseases are due to loss of function of BMP signaling ,2,3, butIn this review, we discuss the important and highly fine-tuned BMP pathway in the context of the endothelium. The major functions of the BMP pathway are first discussed in the high-flow circulatory system, and then emphasis is given to the emerging BMP functions in the low-flow unidirectional lymphatic system. Currently, the role of BMP signaling in the lymphatic system is primarily documented in animal models and human cell cultures. However, it can be expected that, in analogy to the blood vasculature and vascular disease, human lymphatic vascular anomalies resulting from unbalanced BMP signaling are likely to be discovered.In 1965, the activity of a BMP was first reported by M. Urist, who described that a proteinaceous component in demineralized bone extracts can induce ectopic bone formation when implanted under the skin or into the muscle in different laboratory animals . MoreoveWe first give an overall view on the core of the BMP pathway  and subsThe BMPs belong to the family of Transforming Growth Factor β (TGFβ) secreted growth and differentiation factors ,9. As thBMP ligands bind as a dimer to a hetero-oligomeric receptor complex consisting of two different serine/threonine kinase receptors, the type I and type II receptors  Figure . Seven tUpon BMP–receptor binding, the constitutively active type II receptor phosphorylates the serine and threonine residues in the glycine–serine-rich (GS) domain of the type I receptor . This trC. elegans and D. melanogaster, respectively, named “Sma” and “Mother Against Decapentaplegic”. Phosphorylation of the type I receptor results in recruitment of the receptor-regulated SMAD effector proteins or R-SMADs. Depending on the type I receptor that is activated, a different subset of R-SMADs is recruited. Activation of the BMP-specific type I receptors ALK1/2/3/6 phosphorylates the so-called BMP-SMADs SMAD1/5/8. Activation of the TGFβ/Nodal/Activins-SMADs SMAD2/3 results from phosphorylation by the type I receptors ALK4/5/7. However, on some occasions, TGFβ can also activate SMAD1/5/8 complexes [The canonical or SMAD signaling pathways are the most extensively documented cascades . The namomplexes .Receptor-mediated phosphorylation of the C-terminal Ser-X-Ser motif of R-SMAD monomers (pSMAD) triggers activation and complex formation between pSMADs and the common SMAD4. This complex is then translocated into the nucleus where it regulates in association with various co-factors (CoF) context-specific pathway-dependent gene expression  Figure .SMADs are subject to numerous post-translational modifications that regulate their activity and allow other signaling pathways to influence the pathway . An examIn addition to the canonical SMAD-dependent pathways, ligand–receptor activation can also trigger non-canonical intracellular effector phosphorylation. Examples of such non-SMAD pathways include MAPK and Phosphoinositide 3 (PI3) kinases/protein kinase B (AKT) ,20,30 F. These nIn this section, the function of BMP signaling components in the blood vasculature is summarized briefly, owing to excellent recent reviews ,2,3,4,31Blood vasculature—BMP2, -4, -6, -9 and -10 are the BMPs with vascular functions  neonates have dilated collecting and capillary lymphatic vessels, and a reduced number of intraluminal lymphatic valves in collecting vessels. BMP9 inhibits Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (Lyve1) expression through ALK1 in mesenteric lymphatic vessels which promotes the maturation of the mesenteric lymphatic vessels. Moreover, BMP9 induces the expression of Forkhead box protein C2 (Foxc2), Connexin37 (Gja4), Ephrin-b2 (Efnb2) and Neuropilin1 (Nrp1) in an ALK1-dependent manner. These genes are all master genes involved in lymphatic valve formation in mice [FOXC2 causing human lymphatic vascular anomalies [Bmp9 KO neonates, adult Bmp9 KO mice show similar lymphatic capillary and collecting vessel maturation deficits and have inefficient drainage of interstitial fluid [Prospero Homeobox 1 (PROX1) expression through ALK1 in human dermal lymphatic endothelial cells (HDLECs), thereby altering cell cycle-related genes, which leads to a restricted HDLEC cell proliferation. Interestingly, the BMP9-mediated downregulation of PROX1 also results in a trans-differentiation of lymphatic endothelial cells to blood endothelial cells [BMP9 is essential for lymphatic vessel maturation and especially lymphatic valve formation ,34. Bmp9 in mice , and witnomalies . Confirmal fluid . InteresSubileau et al. showed the morphogen properties of BMP9 in lymphangiogenesis in mouse embryonic stem cell differentiation experiments . A low dProx1 expression in zebrafish, which impedes zebrafish lymphatic vessel development. The BMP2-mediated repression of PROX1 was confirmed in HDLEC cultures, similar to the BMP9-mediated downregulation of PROX1 reported by Yoshimatsu et al. [miR-31 and miR-181a in a SMAD-dependent manner. Expression of miR-31 and miR-181a is present in vascular endothelial cells in the cardinal vein, but not in lymphatic endothelial cells that are budding from the cardinal vein. The authors postulated that these miRNAs target Prox1 and thus restrict lymphatic endothelium specification in the cardinal vein. Additionally, co-administration of BMP2 and VEGF-C in a lymphatic differentiation model using mouse embryoid bodies showed that BMP2-SMAD1/5/8 signaling inhibits the normally VEGF-C-mediated lymphatic endothelial cell induction in the periphery of the embryoid body. Interestingly, in the developing embryo, pSMAD1/5/8 activity was detected in vascular endothelial cells in the cardinal vein, although not in the budding lymphatic endothelial cells, similar to the expression of miR-31 and miR-181a. This study shows that the BMP2 function in the lymphatic vasculature is conserved over different vertebrates [In addition to BMP9, BMP2 signaling was shown to negatively regulate lymphatic vessel development in zebrafish and mice in a SMAD- and miRNA-dependent manner . BMP2 gau et al. . Moreovetebrates .Bmp2, Bmp6 and Smad6/7, while cells lining the ceiling express Bmp4 [A recent single-cell (sc)RNAseq study to investigate lymphatic endothelium specialization in mouse lymph nodes revealed that several BMP ligands and target genes of BMP signaling are differentially expressed within the lymphatic endothelium of lymph nodes. For instance, the lymphatic endothelium that lines the floor of the subcapsular sinus expresses ess Bmp4 . This fuBlood vasculature—The pro-angiogenic BMPs BMP2/4/6/7 signal mainly via the ALK2, ALK3 and ALK6 BMP type I receptors, in conjunction with either BMPR2 or ACVRIIs type II receptors in the endothelium. The anti-angiogenic BMPs BMP9/10 induce signaling via ALK1, the most abundant type I receptor in endothelial cells. The ligand–receptor complexes in the blood vasculature are reviewed in [iewed in   but also HDLECs express the BMP type I receptor ACVRL1 encoding ALK1, ACVR2B (ACTRIIB), BMPRII and ENG, and concomitantly that HDLECs respond to BMP9/10 in an ALK1-dependent manner [Bmp9 KO embryos and neonates compatible with the scenario that BMP9 predominantly acts through ALK1 to restrict lymphangiogenesis [t manner . The impogenesis .Smad5, but not of Smad1 or Smad9, resulted in reduced numbers of lymphatic endothelial cells, suggesting that only Smad5 is indispensable in zebrafish lymphangiogenesis [Zebrafish studies have shown the involvement of the type I receptors Alk3/Alk3b and the type II receptors Bmpr2a/Bmpr2b in lymphatic development. Morpholino anti-sense oligonucleotides were used to examine the effects of silencing of these BMP signaling components on lymphatic development. In contrast to the anti-lymphangiogenic effect of Bmp2 in zebrafish , reductiogenesis .Blood vasculature—SMAD1/5/8 activity, monitored by the presence of nuclear pSMAD1/5/8 proteins, has been described in numerous vascular beds in several studies. Moreover, BMP signaling via SMAD1/5 has been shown to be essential for stalk cell identity during sprouting angiogenesis in mouse embryos [SMAD9) seems to function especially during pulmonary vascular homeostasis in adult mice [Smad6/7, Atoh8 encoding atonal basic helix-loop-helix transcription factor 8, Tmem100 encoding transmembrane protein 100, EGFL7 encoding epidermal growth factor-like domain 7 and Id-genes encoding inhibitors of differentiation proteins [miR-21 and miR-27a and suppresses vascular endothelial growth [ embryos . Stalk cult mice  and humault mice ,49, illuproteins ,53,54,55l growth . The nonl growth .Lymphatic vasculature—Many BMP reporter mice have been generated to investigate the transcriptional activity of BMP-SMAD signaling [ignaling ,59,60,61ignaling . Beets eignaling . Mosaic ignaling . It is mMoreover, dermal embryonic lymphatic vessels at E14.5 and E16.5 displayed a prominent and widespread GFP pattern. In postnatal tissues, GFP-positive lymphatic endothelial cells were mainly confined to the valve-forming regions in collecting vessels . This obTmem100—a target gene of the BMP9/ALK1 axis in vascular endothelial cells. Proliferation and apoptosis are unaffected in the lymphatic endothelium. Conversely, endothelial-specific overexpression of TMEM100 results in reduced lymphatic endothelial progenitors, and a small size and number of disorganized lymphatics. This is compatible with a role of TMEM100 in restricting—just like the Notch pathway and putatively  through Notch signaling—the specification of the lymphatic endothelial cell lineage [Interestingly, increased numbers of lymphatic progenitors and enlarged and blood-filled lymphatics are present in an endothelial-specific KO of  lineage . This stThe shaping of BMP morphogen gradients or responses depends on the bioavailability of (tissue-specific) BMP ligand–receptor complexes, and intracellular effectors . In addiBlood vasculature—BMPs are just like other TGFβ members synthesized as large pre-proproteins comprising a signal peptide, a large pro-domain and a mature growth factor domain and then processed as a mature disulfide-linked dimer. For a BMP to become active, dimerization is a prerequisite. Thereto, its pre-proprotein is cleaved by subtilisin-like proprotein convertases, such as Furin; thereafter, the two pro-domains come together to fold and form a covalently bound dimer [nd dimer ,64,65. DThe pro-domain is an important regulator of latency of TGFβ family members, since the pro-domain can block binding to the receptor complex or affect interaction with the extracellular matrix (ECM) through fibronectin or fibrillin assemblies ,66. The Mature BMP7 interacts in mice with the fibrillin2 component of the fibrillin assemblies that are present in the ECM of endothelial cells . Moreove“Mixed” BMPs, achieved by pro-domain heteromerization, are another important diversification of BMP signaling, potentially also in the context of vessels. Recombinant BMP2/5, BMP2/6, BMP2/7 and BMP2b/7 heteromers have been shown to more potently activate the signaling pathway that induces dorsoventral patterning in zebrafish embryos than the respective homodimers ,79. AddiLymphatic vasculature—BMP9 and BMP10 are synthesized as large pro-domain-associated precursors that freely circulate in the blood stream while still being associated with their pro-domains. This association does not hamper receptor binding [ binding , and hen binding ,82. It iBlood vasculature—Binding of BMPs with low type I and/or type II receptor binding affinities can be enhanced by the engagement of a co-receptor in the complex, leading to an increased diversity in ligand–receptor assembly [assembly . Here, wEndoglin (CD105) is a type III accessory receptor abundantly, but not exclusively, expressed by activated vascular endothelial cells ,85, withBetaglycan is another glycoprotein type III accessory receptor which interacts with TGFβ2 but also with BMP2, -4 and 7, enhancing the further downstream pathway. Moreover, it has been shown that betaglycan is essential in endothelial-to-mesenchymal transition (EndMT) in the developing mouse heart through BMP2 signaling enhancement ,89.Another type of co-receptor are the glycosylphosphatidylinositol (GPI)-anchored receptors, called the RGMs. These co-receptors sensitize cells for BMPs present at low concentrations, but such activities have not yet been reported in the endothelium ,91,92.Interestingly, NRPs and vascular endothelial (VE)-cadherin are important co-receptors for vascular endothelial growth factor (VEGF) signaling, which also interact with BMP/TGFβ signaling receptors. NRP1 and NRP2 bind TGFβ, ALK1/5, the type II receptors and betaglycan, affecting TGFβ ligand sensitivity and sprouting angiogenesis ,94,95. VLymphatic vasculature—It is likely that the BMP pathway is tuned in the lymphatic endothelium by similar NRP1-, NRP2- or VE-cadherin-mediated interactions with the BMP receptor complex to those in the blood vasculature (previous paragraph), but hard evidence is still lacking.Blood vasculature—The BMP antagonists  is expressed on endothelial cells and acts as a non-signaling, competitive antagonist of type I receptors such as ALK 1 and -5. Deficiency of BAMBI in mice results in an unusual arterial wall neovascularization that surprisingly mimics features of intra-plaque hemorrhage of advanced atheroma in a mechanical injury model. This is compatible with a role of BAMBI in arterial endothelial cell homeostasis .Xenopus embryos [R-spondins are secreted co-activators of WNT signaling with functions in the vascular  and the  embryos . ConfirmLymphatic vasculature—The above-described BMP antagonists have thus far not been described to modulate BMP signaling in the lymphatic vessel wall.Blood vasculature—The importance of mechano-transduction in the (dynamic) regulation of endothelial phenotypes and features is well appreciated in vessel biology [ biology ,121. Mec biology . Interes biology ,121  in arteries to 0.1–0.6 Pa with low pulsatility in veins [BMP4, Noggin and Grem1, is regulated by flow. For instance, BMP4 is strongly downregulated in laminar shear stress conditions in vascular endothelial cells, whereas it is induced under disturbed flow conditions [Responses of vascular endothelial cells to fluid hemodynamic forces from the blood flow govern the development, physiology and diseases of vessels. These responses already occur during the initial development of the vascular plexus, when hemodynamic mechanical stimuli and growth factors contribute to the patterning and remodeling of the plexus . Indeed,in veins . Interesnditions . Moreovenditions .An intriguing mechanism by which mechanical forces can affect the BMP pathway is how such ligands are made bioavailable. As described in Another mechanism of how mechanical cues tune BMP signaling is through colocalization of integrins and focal adhesion components with BMP receptors and the internalization of the BMP receptors . As mentJunctions in vascular endothelial cells are another site in the cell membrane where mechano-transduction and BMP signaling interact. For instance, the gap junction protein connexin37 has recently been described to be differentially regulated upon mechanical stimulation of SMAD1/5/8 signaling in endothelial cells . AdditioThe luminal primary cilium is a signaling hub that integrates, amongst others, flow-derived signals. The BMP/ALK1 axis is specifically enriched at the primary cilium, and prominent phosphorylation of SMAD1/5/8 is observed at the basal body and along the cilium. Furthermore, FSS increases the physical interaction between ALK1 and endoglin and thereby lowers the effective concentration of BMP9 required for ALK1 activation. The cilium thus sensitizes blood endothelial cells to BMP9 ligands and prevents excessive vascular regression .Interestingly, the intracellular actin cytoskeleton and mechanosensory components of the nucleus also affect BMP signaling output and vice versa. For example, actin-driven filopodia and cytoskeletal rearrangements are vital for the leading tip cell in angiogenic sprouts . BMP6 reLymphatic vasculature—The lymphatic vasculature is a low-flow system (up to 0.6 Pa) with a very high pulsatility [satility . The lymsatility ,140. Colsatility . Moreovesatility .Lymphatic endothelial cells express α9 and β1 integrins. More specifically, α9 integrin is necessary for ECM deposition in the valve-forming region and subsequent valve development . β1 inteGJA4), connexin43 (GJA1) and connexin 47 (GJC2) are produced by lymphatic endothelial cells, with major functions for connexin37 and connexin43 in lymphatic vessel development [GJA4 is induced by PROX1, FOXC2 and flow and induces, in turn, the nuclear factor of activated T cells (NFATC)/calcineurin pathway to promote valve formation in the lymphatic vessel [Several publications reported that connexin37  SMAD6 and SMAD7 provide an intracellular negative feedback mechanism on BMP signaling [ignaling ,162,163 ignaling ,165. Sevignaling . SMAD6 hignaling . Moreoveignaling . These dLymphatic vasculature—Little information is available on the functions of I-SMADs in the lymphatic vasculature, yet SMAD7, SMAD4 and VEGF-D have been correlated with lymphangiogenesis and lymph node metastasis in patients with colon cancer [n cancer . High leBlood vasculature—SMADs do not only form complexes amongst themselves but also engage in physical interactions with a large number of non-SMAD proteins [proteins ,169  or TβRII show that TGFβ signaling has a dual role: it is necessary for proper lymphatic sprouting and network formation in the dermis while restricting lymphatic endothelial cell proliferation. Moreover, TGFβ2 is the critical ligand in these processes in mice and regulates the expression of VEGFR3 and NRP2 in human lymphatic endothelial cell cultures [NR2F2 and SOX18 encoding COUP-II and SRY (sex determining region Y)-box 18 transcription factors, and by repressing PROX1 and LYVE1 expression, respectively [cultures . Given tectively ,176. TheBlood vasculature—The VEGF pathway is an extensively studied pathway with major functions throughout the cardiovascular and lymphatic systems. This pathway regulates processes such as endothelial cell migration, survival, proliferation, permeability, tube formation and metabolism [tabolism . In mammtabolism . VEGF-A tabolism ,179.BMP2/6/9, but also Bmper and Endoglin, are all induced by VEGF in the endothelium, in vitro and in vivo [Vegf-a [Vegf-a in aortic endothelial cells [VEGFR2 in HUVECs [VEGFR2 and promotes VEGFR1 expression [In the circulatory system, numerous cooperation between VEGF signaling and BMP signaling have been reported  and Jagged (JAG) ligands and Notch receptors resulting in proteolytic processing of the Notch receptor and release of the Notch intracellular domain (NICD). The NICD effector translocates into the nucleus where it complexes with Mastermind and Recombination signal binding protein for immunoglobulin kappa J (RBP-J) to activate expression of Notch target genes such as those encoding the basic-helix-loop-helix (bHLH) proteins hairy and enhancer of split-1 (HES1), Hairy/enhancer-of-split related with YRPW motif protein (HEY) 1 and HEY2 [and HEY2 .HEY2 and JAG1 in endothelial cells through binding of pSMAD1/5 to GC-SMAD binding elements (SBE) in their promoter [Hey1/2 [Hey-1/2 and Efnb2 encoding Ephrin B2 in a Notch-independent manner [BMP9 signaling and BMP6 signaling directly regulate the expression of promoter . Moreove [Hey1/2 . Interest manner ,200,201 Jag1 being induced in the endocardium by BMP signaling. JAG1-Notch1 signaling regulates, in turn, Bmp2 expression in the myocardium [The BMP and Notch cascades interact extensively in vascular endothelial cells, amongst which there is early arterial/venous specification in the early mouse embryo, balancing the tip–stalk cell ratio in angiogenic sprouting and heart valve development ,203. Forocardium ,202,203.ocardium .Hes1 [Smad6 expression and thus reduces the BMP responsiveness of endothelial cells and new vessel branch formation [The BMP-pSMAD1/5/8 pathway cooperates also dynamically with Notch signaling in the establishment of a robust stalk cell phenotype during angiogenic sprouting ,204. In Hes1 . HoweverHes1 ,197. WavHes1 . Additioormation .Lymphatic vasculature—It is very probable that there is, just like in the vascular endothelium, a direct cross-talk between BMP and Notch signaling in the lymphatic endothelium. The Notch pathway limits the number of lymphatic endothelial progenitors in the cardinal vein [nal vein , tempersnal vein  and funcnal vein ,208. Intnal vein .The molecular link between BMP and Notch functions in the lymphatic endothelium is still lacking; however, such a missing link may be TMEM100 in lymphatic endothelium specification. TMEM100 is a target gene of the BMP9/ALK1 axis in vascular endothelial cells that is also described in It remains to be investigated whether there are functions for BMP signaling in the intestinal lacteals, the lymphatic capillaries in intestinal villi. Here, DLL4-Notch-mediated signaling promotes the regeneration of intestinal lacteals .Blood vasculature—Canonical and non-canonical WNT signaling pathways play a crucial role in cardiac development, blood vessel development and maturation [turation . The canturation ,212. Thituration .Aggf1 encoding angiogenic factor with G-patch and FHA domains 1, β-catenin-mediated gene expression of NR2F2 or COUP-TFII in the cardinal vein to promote the differentiation of venous endothelial cells. Thus, BMP signaling promotes the venous cell fate by regulating β-catenin, the main intracellular effector of the canonical WNT pathway [WNT and BMP signaling pathways cooperate and attenuate each other in a context- and tissue-dependent manner , and BMP receptor genes in mice causes embryonic lethality, with most prominent defects in mesoderm formation and cardiovascular development. This illustrates that this pathway exerts critical functions during embryogenesis [ogenesis ,2,8. Addogenesis ,2,3,4 , the activin receptor like type 1  or ALK1 (ALK1), and occasionally also in SMAD4 , BMP9  and BMPRII , cause different subtypes of HHT [Gja4 (connexin37), preventing the formation of AVMs in a proliferation-independent but shear stress-regulated manner [Patients with HHT present in specific tissues, such as the lung, brain, gastro-intestinal tract and liver, a paucity in capillaries and arteriovenous shunts/malformations (AVM). This results in a high-blood pressure blood flow from the arteries directly into the thinner-walled, less elastic veins whereby the capillary bed is bypassed. This ultimately results in enlarged blood vessels and severe hemorrhages and epistaxis. HHT is a rare disease with a frequency of about 1 in 6000 . Mutatios of HHT ,224,225.s of HHT ,227. Ints of HHT ,229. Indd manner .BMPR2) [BMPR2 [BMPR2 mutation carriers further developing PAH. In a few patients, mutations have also been reported in ACVRL1, ENG or SMAD9, with SMAD9 confusingly encoding the SMAD8 protein [Patients suffering from PAH (MIM: 178600) are characterized by an increased pressure in the pulmonary artery due to aberrant vascular remodeling of the arteries, ultimately causing heart failure  PAH is aBMPR2) ,234 are ) [BMPR2 .The pene protein . Overall protein ,237,238.CCM genes has been shown to induce EndMT via an exacerbation of BMP6 signaling (MIM: 116860). This gain of function of BMP signaling is a crucial event in the onset and progression of brain cavernomas, with locally increased permeability and hemorrhaging in the central nervous system [Patients with CCM are characterized by cerebral cavernous angiomas. These are rare vascular malformations that may involve any part of the central nervous system. The prevalence of this rare disease is about 0.1% to 0.8% of the population . Loss ofs system ,240.SMAD6 gene. Here, in addition to the occurrence of a bicuspid aortic valve, a dilation of the ascending aorta is present [Patients with BAV are characterized by an aortic valve with two rather than three leaflets. BAV is the most common congenital heart defect, with an estimated prevalence of 0.5 to 2% of the population , and is  present .Aberrant BMP signaling has also been implicated in the disease progression of atherosclerosis in vitro and in vivo. Using several pharmacological inhibitors of BMPs in in vitro and mouse studies, it was shown that BMP signaling regulates endothelial cell activation and cell differentiation in and around the atherosclerotic plaque ,245,246.While atherosclerotic obstruction is the most frequent culprit for peripheral arterial disease, the latter also triggers a rescue response whereby blood flow is redirected through remodeled collateral arteries. BMP-SMAD signaling has been shown to be involved in this shear stress-dependent remodeling response through its downstream mediator muscle segment homeobox 1 (Msx1) .ACVR1 gene encoding the BMP type I receptor ALK2 [The ultrarare autosomal dominant disease FOP  is caused by heterozygous gain-of-function mutations in the tor ALK2 . FOP muttor ALK2 .Lymphatic vasculature—Interestingly, prominent lymphatic anomalies have thus far not been reported in the rare vascular diseases described in KDR, FLT4, NRP2, GATA2) [Although several genetic predisposition factors for lymphedema are also target genes of BMP signaling  ,256,257,, GATA2) ,34, and The growing wealth of single-cell transcriptome data and protein–protein interaction databases, also in lymphatic vascular cells from different niches, will progressively reveal putative additional functions of BMPs in lymphatic endothelium specification and functions. Ligand–receptor pairing between lymphatic vascular cells and cell types in their environment and analysis of target genes of BMPs will facilitate inferring the intercellular communication and BMP-sensitive niche-specific specialization of the lymphatic endothelium. In any case, it will be important to consider the context-dependent regulation of this pathway, which contributes to the subtle variations in functions of the BMP signaling components in different lymphatic endothelial cell types.The first data on human lymphatic malformations and BMPs, together with the reported studies in zebrafish and mice, suggest a potential role for BMP signaling in human lymphatic vessel development and/or maturation. The many examples that are provided in this review on very specific BMP functions and tuning of this pathway in the vascular endothelium and the emerging picture of a cross-talk between BMP signaling and mechanobiology and VEGF, Notch and WNT signaling in the (lymphatic) vasculature can inspire the lymphatic vessel field. Moreover, the recent finding that BMP6 regulates TAZ-Hippo signaling and neo-vessel formation in the vasculature , as well


This paper presents a three-color balance adjustment for color constancy correction. White balancing is a typical adjustment for color constancy in an image, but there are still lighting effects on colors other than white. Cheng et al. proposed multi-color balancing to improve the performance of white balancing by mapping multiple target colors into corresponding ground truth colors. However, there are still three problems that have not been discussed: choosing the number of target colors, selecting target colors, and minimizing error which causes computational complexity to increase. In this paper, we first discuss the number of target colors for multi-color balancing. From our observation, when the number of target colors is greater than or equal to three, the best performance of multi-color balancing in each number of target colors is almost the same regardless of the number of target colors, and it is superior to that of white balancing. Moreover, if the number of target colors is three, multi-color balancing can be performed without any error minimization. Accordingly, we propose three-color balancing. In addition, the combination of three target colors is discussed to achieve color constancy correction. In an experiment, the proposed method not only outperforms white balancing but also has almost the same performance as Cheng’s method with 24 target colors. A change in illumination affects the pixel values of an image taken with an RGB digital camera because the values are determined by spectral information such as the spectra of illumination ,2,3. In Applying white balancing requires a two-step procedure: estimating a white region with remaining lighting effects  and mapping the estimated white region into the ground truth white without lighting effects. Many studies have focused on estimating a source white point in images ,17,18,19In contrast, Cheng et al.  proposedIn this paper, we propose a three-color balance adjustment. This paper has two contributions. The first one is to show that if the combination of three target colors that provides the lowest mean error under three-color balancing is chosen, the three-color balancing will have almost the same performance as Cheng’s method with 24 target colors. The second one is to show that there are multiple appropriate combinations of the three target colors, which can offer a low mean error even under various lighting conditions. From the second, some examples of the combination of three target colors are recommended, which can maintain the performance of three-color balancing under general lighting conditions. As a result, the proposed three-color balancing can more improve color constancy correction than WB. Furthermore, the proposed method gives us a new insight for selecting three target colors in multi-color balancing.In experiments, three-color balancing is demonstrated to have almost the same performance as Cheng’s method with 24 target colors, and it outperforms conventional white balance adjustments. Additionally, the computational complexity was tested under various numbers of target colors, and the proposed method achieved faster processing than Cheng’s method with error minimization algorithms.Here, we summarize how illumination change affects colors in a digital image, and white balancing and Cheng’s multi-color balancing are summarized.On the basis of the Lambertian model , pixel v720] see  2,3]. A. AE(λ), Equation  means thaptation . To mimiBy using white balancing, lighting effects on a white region in an image are accurately corrected if the white region under illumination is correctly estimated. Many studies on color constancy have focused on correctly estimating white regions, and a variety of automatic algorithms are available ,15,17,18White balancing is performed byEquation  is givenor space , and PWBmage IWB . MA withransform .  [n is greater than three, In addition, Cheng et al. proposed a method  for consn chart) . If n isoinverse ,34 is usHowever, as noted by Funt et al. , becauseEquation  is insufEquation  as the iAs well as white balancing, a pixel value corrected by Cheng’s method quations –8)..PCNG is (i)How the number of target colors is decided.(ii)n target colors is selected.How the combination of (iii)How the computational complexity of Cheng’s method is reduced.However, this method still has three problems that have not been discussed:Additionally, as for (iii), if the number of target colors is increased, the computational complexity of Cheng’s method will be increased due to the use of Equation .In this section, we investigate the relationship between the number of target colors and the performance of Cheng’s multi-color balancing. From the investigation, we point out that if the combination of three target colors that offers the lowest mean error in three-color balancing is chosen, the three-color balancing will have almost the same performance as Cheng’s method with 24 target colors. Accordingly, we propose a three-color balance adjustment that maps three target colors into corresponding ground truth colors without minimizing error. Additionally, the selection of three target colors is discussed, and we recommend some example combinations of three target colors, which can be used under general illumination. Finally, the procedure of the proposed method is summarized.In this section, we argue the relationship between the number of target colors and the performance of color constancy correction.n was set to 1 for white balancing, and it was set to 3, 4, 5, and 24 for Cheng’s method. Additionally, corresponding ground truth colors were selected from White balancing with XYZ scaling and Cheng’s multi-color balancing were applied to the images in n target colors were chosen from 24 patches in the color rendition chart. Therefore, the combination number of n target colors was For every combination of target colors, the performance of each adjustment was evaluated by using the mean value of reproduction angular errors for the 24 patches. The reproduction angular error is given byruth one . In this(i)Cheng’s method had a lower minimum mean error than white balancing.(ii)When Moreover, when Equation  is not rLet s colors ,38,39,40When both By applying Equation  to everyThe proposed method is considered as a special case of Cheng’s multi-color balancing. If the number of target colors is three and both Under one light source, the optimal combination of the three target colors has to be selected by testing all of the conceivable combinations, as discussed in We used the ColorChecker dataset prepared by Hemrit et al. , in whicAdditionally, we used the other 51 images from the dataset to evaluate the effectiveness of the combination of three target colors. In From From the figure, the five combinations of three target colors had almost the same result. This means that the effectiveness of three-color balancing can be maintained even when  is replaced with other combinations. Therefore, for example, we recommend  and  from When we assume that three colors selected from 24 colors in (i)Select three ground truth colors from (ii)Prepare three objects in a camera frame, in which each object color corresponds to one of the ground truth colors. Compute three target colors (iii)Apply three-color balancing by using Using  or  as a combination is recommended in step (i), as discussed in We conducted experiments to confirm the effectiveness of the proposed method.In this experiment, the effectiveness of three-color balancing with the recommended combinations was demonstrated by using various colors and lighting conditions, where these experimental conditions were different from those in Representative RGB values were computed as the mean-pixel value of a color region in The proposed method was applied to the input images, and it was compared with white balancing and Cheng’s method (Cheng (1–24)), where XYZ scaling (WB (XYZ)) and von Kries’s model (WB (von Kries)) were applied as white balance adjustments. Target colors were selected from the color rendition chart indexed from 1 to 24 in In In Cheng’s method, all of the 24 patches in a color rendition chart are used as target colors. Additionally, when Equation . In contEquation  is not rTo evaluate the complexity, we implemented white balancing with XYZ scaling, three-color balancing, and Cheng’s method on a computer with a 3.6 GHz processor and a main memory of 16 Gbytes see .Note that the runtime of the equations shown in As shown in In the proposed three-color balancing, when Equation . If T anficiency . Figure (i)If the product of eigenvalues is small, the mean value of reproduction errors will be high.(ii)There are many combinations of three target colors at which three-color balancing results in a high error.(iii)Analysis based on eigenvalues enables us to select three target colors without a large dataset as in In a manner like n target colors and the selection of three target colors for the proposed three-color balancing. When the number of target colors is over three, the best performance of color constancy correction is almost the same regardless of n. Additionally, for three-color balancing, a combination of three target colors that enables us to achieve a lower reproduction error was determined with a large dataset. Moreover, no algorithms for minimizing error are required to use the proposed method, and this contributes to reducing computational complexity. Experimental results indicated that the proposed three-color balancing did not only outperform conventional white balancing but also had almost the same performance as multi-color balancing with 24 target colors. In this paper, we proposed a three-color balance adjustment for color constancy. While conventional white balancing cannot perfectly adjust colors other than white, multi-color balancing including three-color balancing improves color constancy correction by using multiple colors. Additionally, this paper presented the choice of the number of


Diabetic retinopathy (DR), a sight-threatening neurovasculopathy, is the leading cause of blindness in working-aged adults , known as “trans-signaling” , is one of the major mediators of retinal vascular inflammation associated with DR . Anti-IL6 and anti-IL6R therapeutic strategies globally block IL-6 signaling, essentially targeting both classical and trans-signaling pathways. Tocilizumab, an IL-6 receptor-inhibiting monoclonal antibody, is useful in the treatment of various autoimmune and inflammatory conditions, notably rheumatoid arthritis  dysfunction and increased vascular permeability, allowing extravasation of plasma proteins into the interstitium  is a natural inhibitor of IL-6 trans-signaling . IL-6 receptor associates with the ubiquitously expressed protein gp130, initiating dimerization and intracellular signaling. L-gp130 is a designer protein in which the entire extracellular portion of gp130 is replaced by the leucine zipper of the Jun protein for constitutive dimerization and activation. Thus, L-gp130 protein can be used for permanent gp130 activation to mimic constitutive IL-6 signaling in cells (Stuhlmann-Laeisz et al., in vivo (Rabe et al., Transgenic mice that constitutively overexpress sgp130Fc are valuable resources to selectively block IL-6 trans-signaling Increasing evidence suggests that the IL-6 pathway plays a prominent role in the pathogenesis of DR. The complex IL-6 receptor system allows for multiple signaling modalities, including classical signaling and trans-signaling. Classical signaling is critical for the regenerative or anti-inflammatory activities of IL-6, while recent studies have demonstrated that IL-6 trans-signaling is primarily pro-inflammatory. In DR, IL-6 trans-signaling mediates barrier disruption in retinal endothelial cells, and blockade of this pathway maintained normal endothelial barrier function. Selective inhibition of IL-6 trans-signaling with sgp130Fc also suppressed ocular inflammation and oxidative stress in a mouse model of DR. These findings indicate that a pathway primarily driven by IL-6 + soluble IL-6R contributes to vascular inflammation in the diabetic retina. Therefore, inhibiting only the trans-signaling pathway of IL-6 will likely be therapeutically superior to a complete IL-6 blockade, because important physiologic functions of IL-6 classical signaling will remain intact. An emerging challenge is identifying means of targeting this inflammatory pathway, as well as determining which DR patients may benefit most from therapies blocking IL-6 trans-signaling. The selective inhibition of IL-6 trans-signaling using the sgp130Fc fusion protein is in clinical trials for the treatment of several inflammatory diseases (Rose-John, The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The results of the mediating effect model show that the more frequently women use the internet, the lower their fertility intentions, and the less they agree with Chinese traditional gender roles, which are “men work outside to support the family while women stay at home to take care of the family”. These findings have implications in formulating public policies aimed at increasing the fertility rate; that is, it is not enough to increase women’s fertility intentions under China’s universal two-child policy. Moreover, public policy formulators need to consider gender role attitudes and the influence of the internet as a method for dissemination of information.The purpose of this study is to verify the influence of internet usage frequency on women’s fertility intentions and to examine the mediating effects of gender role attitudes, under the influence of internet usage frequency that affects women’s fertility intentions, combined with the specific Chinese cultural context. A cross-sectional secondary data analysis was conducted using a sample of 3113 women of childbearing age in the Chinese General Social Survey in 2017 (CGSS2017). The results of the negative binomial regression model showed that, under the premise of controlling individual characteristic variables, the higher the frequency of internet usage, the lower the fertility intention ( The implementation of the Chinese universal two-child policy is seen as an important measure to ease the pressures of a reducing labor force and population aging . HoweverThese profound social and economic implications, as a result of the internet, have been underlined by social science scholars throughout the years . RelatedAs a pervasive innovation that affects all realms of society, internet usage likely affects female fertility intentions. On the one hand, internet usage has reduced the cost of accessing information about contraceptives, and the possible consequences of choosing whether or not to become a parent  . On the In addition to the direct effects, the effects of internet usage on women’s fertility intentions can be moderated by gender roles in specific social and cultural contexts in China. China’s traditional concepts of gender roles can imprison women in families, who are responsible for housework and child rearing. By these concepts, fertility intentions are generally higher. However, the popularization of internet usage has awakened women’s independence, reducing their intentions to bear children. To be specific, internet usage narrows the differences between male and females in regards to resources, skills, knowledge, etc., and provides women with more job opportunities . With anBased on the above considerations, this article combines the data from the Chinese General Social Survey in 2017 (CGSS2017) to systematically investigate the impact of internet usage on women’s fertility intentions. Compared to the existing literature, the marginal contributions of this article are as follows: (1) many of the previous studies are family-centered, and women with childbirth intentions and behaviors are often regarded as passive recipients of policy implementations or contraceptive services. This article overcomes these limitations and explores the theoretical mechanisms on how the frequency of internet usage affects a female’s fertility intentions. (2) Compared with Francesco (2019), who analyzed the effects of internet usage frequency and individual fertility intentions from the perspective of family–work conflicts, this article takes another approach, by introducing gender role attitudes into the research framework of women’s fertility intentions and internet usage frequency, and researches the factors affecting individual fertility intentions. (3) This work observes the problem of internet usage frequency and women’s fertility intentions in the Chinese social–cultural context, clarifies the mediating effects of gender role attitudes under the influence of internet usage frequency on women’s fertility intentions, and provides useful support for exploring solutions to the low fertility dilemma as a result of internet usage.The data in this paper were derived from the Chinese General Social Survey in 2017 (CGSS2017), which is a national representative continuous survey project, conducted jointly by the Hong Kong University of Science and Technology and the National Survey Research Center (NSRC) at Renmin University of China. CGSS2017 covers 31 provinces  in mainland China. It adopts a multi-stage stratified random sampling method, which is conducted through individual interviews and structured questionnaire surveys. The CGSS2017 is characterized by full coverage and representativeness, and the questionnaire has good authenticity and reliability. To ensure the reliability of the empirical analysis, this paper processed the original data as follows: (1) to highlight the dominant position of women as the direct bearers of fertility intentions and fertility behaviors, we only retained the samples of women. (2) We limited the samples to women aged 18–50 years old; that is, we only kept samples of women of childbearing age. Following the exclusion of samples with missing values and invalid data resulting from refusal to answer, we finally obtained 3113 valid samples. The statistical software used in this study is STATA version 15.0 software . The current study is approved by Chinese General Social Survey (CGSS).iFI obeys the Poisson distribution with parameter iλ, then the conditional expectation function of iFI is:Since the dependent variable (fertility intention) in this article is a non-negative discrete random variable, which does not meet the basic assumption of a normal distribution of classical linear regression, we used Poisson regression or negative binomial regression in the counting model for parameter estimation. Assuming that female individual fertility intention iμ ≡ exp  > 0 is a random variable. Based on this, let iFI represent the women’s fertility intention and ifi still follow the Poisson distribution. Given ix and iν, the conditional density function of iFI is as follows: The limitation of Poisson regression is that it requires the variance of the dependent variable to be roughly equal to the expected value; that is, “equal dispersion”. After testing the data, we found that the dependent variable did not meet the requirements of “equal dispersion”, so it was more scientific to use negative binomial regression. The usual approach is to add a term to the logarithmic expression of the conditional expectation function of Poisson regression. By introducing unobservable effects that affect women’s fertility intentions, the Poisson model can be extended as follows:E(iFI|ix) = iμ = exp(ix’β), ix  refers to various influencing factors of the fertility intention iFI. According to the setting of this article, the specific model as follows:Among them, iInt is an independent variable, representing the frequency of individual internet usage. iX stands for the set of control variables, including age, marital status, education level, health status, family income level, social interaction, socioeconomic status, and other variables. In Formula (4), This article used the individual’s ideal number of children as an indicator for the measurement of the explained variable (fertility intention). The survey item on this variable in the CGSS2017 data was: “Regardless of policy restrictions, how many children do you want to have?” This article selected this item as a substitute variable for the individual’s intention to bear children ,20.The independent variable of this article was the frequency of internet usage. The survey item for this variable in CGSS2017 data was “How often did you use internet media (including mobile internet access) in the past year?” Based on the respondents’ answers, internet usage was measured with a dummy variable (1 for frequently and 0 for infrequently).The intermediary variable in this article was gender role attitudes. CGSS2017 asked the interviewees about their views on gender role attitudes from the following two aspects: (1) “GRA1—Do you agree with the view that men put career first and women put family first?” (2) “GRA2—Do you agree that the responsibility of the husband is to make money, and the responsibility of the woman is to take care of the family?” Based on the respondents’ answers, the options ranged from “absolutely disagree” (=1), “relatively disagree” (=2), “neutral” (=3), “relatively agree” (=4), “absolutely agree” (=5).The control variables of individual characteristics were set as follows: (1) Age referred to the actual age of the interviewee. In order to test the nonlinear effect of age, we also added the age squared term. (2) Marital status was measured with a dummy variable (1 for married and 0 for unmarried). (3) The variable options of health status included “badly healthy”, “less healthy”, “normal”, “fairly healthy”, and “extremely healthy”. (4) Educational levels were expressed as “middle school and below”, “high school/vocational school”, and “college and above”. (5) The household income level was measured by the question: “What do you think of your income level in the locality?” with response categories “far below average”, “below average”, “average”, “above average”, and “much above average”. (6) As for Individual social interactions, according to the frequency of the social interactions of the interviewees, the rating ranged from “never”, “rarely”, “sometimes”, “often”, and “frequently”.To more rigorously verify the rationality of internet usage frequency affecting fertility intentions and the mechanisms, this paper drew on Baron and Kenny , IacobuciInt is the dependent variable, representing the frequency of individual internet usage. iFI is the independent variable, which represents the interviewees’ fertility intentions. iGRA is the intermediary variable, and ε1–ε3 denotes the error term. iX is a set of control variables, including the interviewee’s age, marital status, education level, health status, family income level, social interaction, and socioeconomic status.0: ab = 0 holds [a and b, respectively, MediationZ is a normal distribution, so we can test the significance of the mediating effect. At the 0.05 significance level, if To confirm whether the significant mediation effect exists, we can test whether the null hypothesis of H 0 holds . The medThe econometric model in this article may also have endogenous problems. Access to the internet is likely correlated with many unobservable determinants of fertility intentions , which may confound the main relationship of interest; that is, whether internet usage frequency is affected by fertility intention. Another problem that needs to be solved is two-way causality. Women with higher fertility intentions have higher demands for effective parenting information, which leads to higher frequencies of network search behaviors. To address these concerns regarding endogeneity, the article employed an IV approach that exploited the average rate of internet usage across the province. The specific calculation method of this variable is that the average internet usage rate of the province is equal to the sum of the internet usage frequency of the province samples, divided by the number of province samples. The reason is that the average internet usage rate of a province has a strong correlation with the internet usage frequency of respondents. However, no evidence shows that the average internet usage rate in a province is related to an interviewee’s fertility intention. A descriptive analysis using means, standard deviations, frequencies, and percentages was computed to explain the samples and variables. A negative binomial regression model was performed to verify the influence of internet usage frequency on women’s fertility intentions after controlling individual characteristic variables. We used a mediating effects model to identify the mediating effects of the gender role attitudes under the influence of internet usage frequency that affects women’s fertility intentions.n = 2179, 70.00%). The majority of women had a desire to have two children . The majority of participants were married  and reported good health status . A minority of the participants had a high school/vocational school  or college degree above .This article first used negative binomial regression to investigate the influence of internet usage frequency on women’s fertility intentions. The benchmark regression results are shown in p = 0.0000). The second-stage regression results showed that the frequency of internet usage had a significant negative impact on women’s fertility intentions ; this result is still robust after adding the control variables, and is basically consistent with the negative binomial regression results. Therefore, after eliminating endogeneity, the results are robust and reliable.To solve the problem of endogeneity, this paper applied the two-stage least square method for quantitative analysis. The results are shown in To analyze the mediating effects of gender role attitudes, we used stepwise regression. First, we tested the influence of internet usage frequency on women’s fertility intentions, and the results are consistent with the results in Based on CGSS2017 data, this article uses negative binomial regression to verify the influence of internet usage frequency on women’s fertility intentions. The results show that the frequency of internet usage has a significant negative effect on women’s fertility intentions after controlling for other variables that might affect their fertility intentions. That is, the higher the frequency of internet usage, the lower the fertility intentions of women. China’s traditional concept of gender division of labor imprisons women more so in families, who are responsible for family production and child rearing. However, the popularization/application of the internet has brought about changes in women’s values, improving their economic status. More and more women become unwilling to play the roles of bearing and rearing children; consequently, this reduces women’s fertility intentions.Our research combines the traditional Chinese cultural context, with particular emphasis on the mediating effects of gender role attitudes in the frequency of internet usage affecting women’s fertility intentions, confirming that the higher the frequency of internet usage, the weaker the consciousness of the traditional Chinese gender role attitudes , and the lower the fertility intentions. This suggests that individual behaviors are influenced by their values. As the theory of moral rationality put forward by Duncan and Edwards shows, individuals do not simply respond to economic or policy incentives but are impacted by their knowledge, values, and ideals ,26. InteThe results of the control variables in econometric benchmark regression show that the higher the education levels of women, the lower the fertility intentions. According to the theory of human capital, improving education, and the abilities that empower women to directly participate in the labor market, have freed more women from raising children, allowing them to pursue their own careers and ideals. This leads to a decrease in their fertility intentions. Family economic status also has a significant impact on women’s fertility intentions. According to the theoretical model of new family economics, fertility decision-making is a function of personal preference and childcare costs under the family income limitation ,29. HighThis article has important implications for policy-makers intending to improve women’s fertility intentions and the fertility rate. From China’s experience, increasing the female fertility rate cannot only rely on a single policy that relaxes the number of births. The negative effects of individual internet usage frequency on childbirth intentions should also be an important factor in evaluating and predicting the effects of fertility policy adjustments. In the process of formulating fertility incentive-related supporting policies, it is also necessary to optimize governance in combination with the network environment to encourage fertility intentions, to better achieve the expected effects of the fertility policy. For example, the government should build a fertility network information service platform to provide high-quality fertility information services to effectively meet women’s needs, to access parenting information and ease anxiety surrounding women’s fertility. Moreover, when investigating the influence of internet usage on fertility intentions, it is necessary to pay special attention to the roles of individual values. To increase women’s fertility intentions, it is also necessary to optimize the division of roles within the family so that women can better balance work and family. Specific measures include encouraging men to shoulder more family responsibilities, and encouraging society to bear part of the direct costs  and indirect costs  of raising children.The main limitations of the study are as follows: 1) this article focuses on the research surrounding women’s fertility intentions, but there may be a gap between the actual number and the ideal number of births and the fertility intentions may not necessarily be reflected in the birth behavior. However, the results of this study can still be considered valid because the intention is an important prerequisite for follow-up behavior ,31. MoreInternet usage has a profound impact on personal behavior and decision-making. In China, internet usage has profoundly changed the traditional gender concept of “men work outside to support the family while women stay at home to take care of the family”, and further reduced women’s fertility intentions. How to deal with low fertility intentions caused by internet usage is a critical issue. The government should regard the negative effects of individual internet use on fertility intentions as an important consideration in evaluating and predicting the effects of fertility policy adjustments. Furthermore, future public policies need to effectively balance family role divisions between men and women, i.e., to highlight more favorable work–family balances for women.


Arabidopsis thaliana) has discovered key peptide signaling pathways, transcription factors, and polarity components that together drive proper stomatal development and patterning. In this review, we focus on recent findings that have revealed co-option of peptide-receptor kinase signaling modules—utilized for diverse developmental processes and immune response. We further discuss an emerging connection between extrinsic signaling and intrinsic polarity modules. These findings have further enlightened our understanding of this fascinating developmental process.Stomata are small pores on the surface of land plants that facilitate gas exchange for photosynthesis while minimizing water loss. The function of stomata is pivotal for plant growth and survival. Intensive research on the model plant Arabidopsis ( Co-opted peptide-receptor kinase signaling modules and intrinsic polarity cues regulate stomatal development. Stomata are turgor-driven microscopic valves in the epidermis of aerial regions of land plants. The controlled opening and closing of the stomatal pores is essential to the regulation of gas exchange and water loss by the plant . In addiThe genetic control of stomatal development requires the consecutive activity of three basic helix-loop-helix (bHLH) transcription factors (TFs), SPEECHLESS (SPCH), MUTE, and FAMA, which drive stomatal initiation, proliferation, and differentiation, respectively . These TSince the discovery of ERf and TMM as a co-receptor controlling stomatal development, various upstream and downstream components have been identified, aiding in the formation of specific peptide–receptor signaling modules and signal transduction.These peptide–receptor signaling modules can act in both paracrine and autocrine manners, dependent on the developmental stage of the respective stomatal cell.The peptide–receptor signaling modules have been co-opted to regulate arrays of developmental programs and intersect with immunity and environmental response.The interplay of extrinsic signals and intrinsic polarity cues, that provide spatial information during cell division, are still not fully understood. However, the discovery of novel polarity proteins and regulatory mechanisms are now unravelling a molecular intersection of different protein modules during stomatal development.ER, which is highly expressed in protodermal cells, primarily acts to repress asymmetric entry division of MMCs, reflected by the increased number of ACDs in the er single mutant. Meanwhile, both ERL1 and ERL2 subsequently function to prevent M differentiation. In the erl1 erl2 double mutant, Ms prematurely differentiate into GMCs, underlining their important role in maintaining the amplifying potential of Ms  domain play key roles in numerous aspects of plant development and immune response . Stomataal of Ms . Severe tmm mutants show altered stomatal patterning reminiscent of er erl1 erl2 triple mutants, i.e. clustered and numerous stomata in the epidermis of cotyledons and leaves , TOO MANY MOUTHS (TMM), lacks the characteristic intracellular kinase domain of LRR–RLKs . tmm mutd leaves . However stomata . Furtherc manner .er-family members and tmm into consideration, one might wonder about their genetic and molecular relationships. Initial genetic evidence has hinted that these proteins contribute in combination to determine stomatal-lineage cell fate, possibly through dimerization /EPF-LIKE (EPFL), has been identified as a regulator of stomatal development . The preEPF2 is expressed in early stomatal lineage precursors, MMCs, and early Ms  signal downregulating SPCH activity. This negative feedback loop prevents surrounding protodermal cells from entering the stomatal lineage , which predicts the future division plane at the opposing end of the future M, showed that its distribution is flawed in both  mutants . Incorre present . Since tERL1 expression, while at the same time ERL1 perceives EPF1 signal to inhibit MUTE activity, creating a negative feedback-loop ensuring the presence of the right amount of MUTE for GC differentiation. This delicate balance between transcriptional regulation and signal transduction can prevent extra symmetric divisions of Ms and GMCs, and an impaired signal transduction by the tmm mutation results in stomatal pairwise clustering, independent of known polarity contributors /MONOPTEROS (MP), which directly binds to auxin-responsive elements within the promoter region of EPFL9/STOMAGEN to repress its expression  positively regulates stomatal development . Express density , suggest density , 2015. Tpression . ConsistThe competitive binding of these peptide ligands with different activities raises the question as to which mode ERf receptor kinase responds to the individual peptides at the subcellular level. A recent study combining genetic, pharmacological, and live cell imaging analyses revealed new insights into the initial subcellular behaviors of the receptor ERL1 upon ligand perception. EPF1, which activates the inhibitory stomatal signaling cascade, triggers TMM-dependent ERL1 internalization into the intraluminal vesicles of multi-vesicular bodies/late endosomes for subsequent vacuolar degradation . ConversCLE9 accumulated highly and specifically in mute scrm-D seedlings, in which the epidermis was solely composed of Ms  peptide family of post-translationally modified dodecapeptides regulate a wide range of biological processes in stem cell homeostasis, as well as in response to phytohormone signaling . The poted of Ms . More reed of Ms . In the ed of Ms . There, ed of Ms . Since tCLE9 and the cytokinin signaling effector gene ARABIDOPSIS RESPONSE REGULATOR16 (ARR16), a M-expressed gene acting as negative regulator for the latter, are directly induced by SPCH in Ms , which bridges the further downstream signal transduction . OriginaPKKK YDA . Thus, uPKKK YDA .scrm-D mutants (which possesses a KRAAM–to-KHAAM amino-acid substitution). Intriguingly, while the putative MAPK docking motif is highly conserved among vascular and nonvascular plants, the KRAAM motif can only be found in SCRM, SCRM2, and their orthologs. This has led to the hypothesis that, while MPK3/6 regulates myriads of developmental, environmental and immunity responses (Formation of the EPFs–ERfs–TMM–SERKs (-BSKs) ligand–receptor signaling complex activates the downstream MAPK cascade, and this activation leads to the degradation of each bHLH TF module . Consequesponses , distincesponses .mpk1 mutant epidermis undergoes asymmetric entry division. However, Ms occasionally fail to differentiate, resulting in rose-petal like SLGC clusters, reminiscent of the mute mutant and EPF1 overexpression phenotypes  controls the phosphorylation status of MAPKs within stomatal precursors downstream of the MAPKKK YDA . mpk1 muenotypes . MUTE ex in mpk1 . These fpp2a mutants or through pharmacological impairment, suppresses stomatal production, indicative of a positive role of PP2A during stomatal development. Furthermore, PP2A-A subunits directly bind SPCH in vitro, suggesting that PP2A may function to regulate the phosphorylation-dependent equilibrium of SPCH protein. Future studies will hopefully illuminate how PP2A activity as well as its regulation is integrated within the ER–EPF2 receptor–peptide signaling module.In addition to MKP1, a recent work revealed that subunits A1, A2, and A3 of the PP2A phosphatase promote stomatal development . Alteratbasl mutants, a high proportion of symmetric cell divisions can be observed in MMCs. Furthermore, a domain analysis of BASL identified its C-terminal region as a prerequisite to direct polarized accumulation at the cell periphery, which was sufficient for polar cell growth  family . One modPolarity proteins from the POLAR LOCALIZATION DURING ASYMMETRIC DIVISION AND REDISTRIBUTION (POLAR) family function at the cortical site distal to the future division plane of premitotic MMCs and Ms in a BASL-dependent manner . POLAR fThe intensive localization analyses of BASL-YDA-MPK3/6 as well as BASL-POLAR-BIN2 modules suggest that their ability to polarize and to form a complex in vivo emanates from BASL’s ability to self-organize in a polar manner upon phosphorylation by the YDA-MPK3/6 cascade . This feWith respect to a potential signal gradient during stomatal development, a recent publication identified new MAPK substrates, which regulate stomatal production in a positive manner . The autER was originally described as a gene regulating inflorescence architecture, found as a spontaneous mutation in the commonly used accession Landsberg erecta , grass species (Brachypodium dystachyon), and moss (Physcomitrella patens), reveal new exciting insights . For example, what molecular mechanisms connect external signaling factors with the intrinsic polarity cues? And how, through the evolution of land plants, have different plant species co-opted and re-wired these signaling and polarity modules to achieve specific stomatal patterns optimized to their ecological niche? Recent studies in monocots, including rice (insights .The ability of plants to regulate themselves to the atmospheric balance and water cycles of our planet is dependent on the precise control of stomatal development and distribution. Since key regulators of stomata development are conserved in grass species and most land plants see also , deciphePlease see


Accruing evidence suggests that astrocytic Ca2+ signals play important roles in both seizures and epilepsy, and this review aims to highlight the current knowledge of the roles of this central astrocytic signaling mechanism in ictogenesis and epileptogenesis.Epilepsy is one of the most common neurological disorders – estimated to affect at least 65 million worldwide. Most of the epilepsy research has so far focused on how to dampen neuronal discharges and to explain how changes in intrinsic neuronal activity or network function cause seizures. As a result, pharmacological therapy has largely been limited to symptomatic treatment targeted at neurons. Given the expanding spectrum of functions ascribed to the non-neuronal constituents of the brain, in both physiological brain function and in brain disorders, it is natural to closely consider the roles of astrocytes in epilepsy. It is now widely accepted that astrocytes are key controllers of the composition of the extracellular fluids, and may directly interact with neurons by releasing gliotransmitters. A central tenet is that astrocytic intracellular Ca Epilepsy is one of the most common neurological disorders – estimated to affect around 1% of the world’s population . It is aThere is a striking lack of knowledge of the pathophysiological cellular mechanisms at play in epilepsy. For instance, the process transforming normal brain matter to a focus for epileptic seizures – the process of epileptogenesis – is not well understood. Also, the central question of what sets in motion an epileptic seizure – ictogenesis – remains unanswered. Most of the epilepsy research has so far focused on how to dampen neuronal discharges and to explain how changes in intrinsic neuronal activity or neuronal network function cause seizures. As a result, pharmacological therapy has been limited to symptomatic treatment aiming at neuronal targets. Given the expanding spectrum of roles ascribed to the non-neuronal constituents of the brain, it is natural to take a closer look at astrocytes as potential targets for epilepsy treatment.+ levels .Astrocytes are critical homeostatic controllers of extracellular glutamate and K+ levels . Numerou+ levels  and that+ levels . Increas+ levels . Notably+ levels . A centrchanisms . Glutama2+ signaling has been demonstrated in seizures and in epileptic tissue, potentially affecting both the homeostatic functions and signaling functions of astrocytes. These downstream mechanisms are largely speculative in the context of epilepsy but reflect the knowledge of roles of astrocytic Ca2+ signaling in physiology. Here, we discuss the relatively limited body of studies directly assessing astrocytic Ca2+ signaling in epilepsy, and briefly discuss potential downstream effects  in nearby neurons that were unaffected by application of the neuronal sodium channel blocker tetrodotoxin  in neurons. These findings proposed a role for astrocytes in synchronizing neuronal activity and contributing to seizure generation  receptor in the endoplasmic reticulum, of which the isoform 2 (IP3R2) is thought to be the key functional IP3 receptor in astrocytes  and subsequent release of Ca2+ from the endoplasmic reticulum via IP3R2 as discussed in “Astrocyte Ca2 + signaling and Ictogenesis” (2+ dependent glutamate release from astrocytes (2+ transients reemerge during epileptogenesis along with an increase in glutamate uptake (2+ mediated gliotransmission.The degree, development and underlying mechanisms involved in aberrant Cadynamics , could bgenesis” . Astrocytrocytes . While atrocytes , the rectrocytes , 2003, ae uptake . This re2+ activated anion channels, and increased GABA release could hence be a downstream effect of increased Ca2+ signaling in reactive astrocytes (Apart from these perturbations in glutamate dynamics, it has been shown that reactive astrocytes exhibit a tonic release of GABA, presumably through Bestrophin-1 channels . Bestroptrocytes . In supptrocytes . Potenti2+ signaling has been suggested to be involved in homeostatic mechanisms of astrocytes. These mechanisms could be important downstream effects of astrocytic Ca2+ dyshomeostasis in epileptic tissue, but these effects are so far rudimentarily investigated in epilepsy.Moreover, as mentioned in “Ictogenesis” astrocytic Ca+ from the extracellular space through the process of K+ spatial buffering (2+ signals to propagate from cell to cell, at least during pathological conditions like seizure activity (2+ waves could play a role in neuronal synchronization and seizure generation. Potentially a loss of astrocytic gap junctions as seen in epileptic tissue, may be a compensatory mechanism to prevent intercellular spread of astrocytic Ca2+ waves. Even so, to the best of our knowledge, no direct study of astrocytic Ca2+ signaling in gap junction deficient mice has been performed.Loss of astrocytic gap junction coupling has been shown to occur during early epileptogenesis in experimental models of mTLE and in specimens of resected hippocampi from patients with mTLE . It is buffering . Notablyactivity . It is t2+ dynamics (2+ signaling in astrocytic endfeet, as we demonstrated in 2+ dependent proteases like calpain (Loss of the highly concentrated expression of key membrane channels in astrocytic endfoot processes, i.e., loss of astrocyte polarization, is another pathological hallmark, which could be a consequence of perturbed glial Cadynamics . For insdynamics , and in dynamics . It is p calpain , that sh calpain .+ handling and resultant neuronal hyperexcitation due to the role of Kir4.1 in K+ homeostasis (Even though the evidence is indirect, it has been suggested that this loss of astrocyte endfoot polarization could contribute to epileptogenesis and hyperexcitation . Notablyeostasis .2+ signaling in ictogenesis and epileptogenesis. These terms are used to describe two different features of epilepsy, but do not necessarily imply two separate processes, as mechanisms crucial in ictogenesis could also be an integral part of epileptogenesis, or vice versa. While we often associate astrocytic dysfunction in epileptogenesis with the appearance of reactive astrogliosis (ictogenesis seems typically to be used when studying the interplay between neurons and astrocytes independent of pre-existing tissue pathology. Therefore, we may overlook the fact that ictogenesis most often would occur in tissue that has undergone pathological transformation typical for epileptogenesis, i.e., not normal, healthy tissue. On the other hand, epileptogenesis comprises many pathological changes beyond reactive astrogliosis, like alterations in transcriptional regulation, morphological, biochemical, metabolic and physiological remodeling ultimately resulting in gain or loss of function (Here we have discussed the role of astrocyte Caogliosis , the terfunction .2+ signals are today considered a main readout of astrocytic activity and there are reasons to believe that they play important roles in epilepsy. Evidence suggests that such signals are neither necessary nor sufficient to maintain epileptiform activity, but rather should be seen as modulators of the pathophysiological process. The literature directly investigating the role of astrocytic Ca2+ signaling in epilepsy is still sparse and at some points contradictory, and for most proposed mechanisms only a small subset of the signaling pathways involved are identified. A major challenge will be to disentangle the potentially beneficial from detrimental consequences of the different modes of astrocyte Ca2+ signaling in reactive astrogliosis. It is even probable that astrocyte Ca2+ signaling may carry different roles in the large variety of epileptic entities. To decipher the roles of astrocyte Ca2+ signaling in epilepsy, next steps should include a rigorous study of the mechanisms mentioned above in vivo in adult mice, leveraging new developments in both imaging and genetics, with the aim of identifying promising targets for future pharmacological therapy of epilepsy.Astrocytic CaKH and RE reviewed the literature, conceptualized the manuscript, and wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Along with playing vital roles in pathogen exclusion and immune system priming, the upper airways (UAs) and their microbiota are essential for myriad physiological functions such as conditioning and transferring inhaled air. Dysbiosis, a microbial imbalance, is linked with various diseases and significantly impedes the quality of one’s life. Daily inhaled exposures and/or underlying conditions contribute to adverse changes to the UA microbiota. Such variations in the microbial community exacerbate UA and pulmonary disorders via modulating inflammatory and immune pathways. Hence, exploring the UA microbiota’s role in maintaining homeostasis is imperative. The microbial composition and subsequent relationship with airborne exposures, inflammation, and disease are crucial for strategizing innovating UA diagnostics and therapeutics. The development of a healthy UA microbiota early in life contributes to normal respiratory development and function in the succeeding years. Although different UA cavities present a unique microbial profile, geriatrics have similar microbes across their UAs. This lost community segregation may contribute to inflammation and disease, as it stimulates disadvantageous microbial–microbial and microbial–host interactions. Varying inflammatory profiles are associated with specific microbial compositions, while the same is true for many disease conditions and environmental exposures. A shift in the microbial composition is also detected upon the administration of numerous therapeutics, highlighting other beneficial and adverse side effects. This review examines the role of the UA microbiota in achieving homeostasis, and the impact on the UAs of environmental airborne pollutants, inflammation, and disease. The upper airways (UAs) compose the respiratory system’s primary elements to conduct and process air to the lungs . An averSeveral environmental factors and/or pre-existing conditions contribute to UA dysbiosis. This review examines the role of the UA microbiota in achieving homeostasis, and the impact of environmental airborne pollutants, inflammation, and disease on the UAs and their symbionts. It is also important to note that age, weight, and diet are also important factors for microbiota composition, diversity, and stability .The UAs are defined as the nasal cavity, sinuses, nasopharynx, oropharynx, and laryngopharynx , all of The nasal cavity is constantly interacting with the external environment ,20. It iLocated adjacent to the nasal vestibule is the middle meatus ,23. ThisProteobacteria, Firmicutes, and Bacteroidetes . Cl. Cl58]. S. aureus by causing the expression of more pathogenic profiles and the development of immune-evasion strategies [Staphylococcus, Planococcaceae, and Lactococcus were detected in the nasal microbiota of individuals with neutrophilia [Staphylococcus, Rothia, and Enterococcus within the nasal microbiota [Propionibacterium can produce bacteriocin, an antimicrobial peptide (AMP) that mitigates the inflammation associated with the overgrowth of pathogenic bacteria [The inflammatory immune response in the UAs is one of the multiple players, including cells such as granulocytes, lymphocytes, and antigen-presenting cells (APCs) ,58. Neutrategies ,60. Aparrategies ,60,61. Srophilia . In addirophilia . Lymphocrophilia . A high crobiota . Conversbacteria .Moraxella [Staphylococcus, Veillonella, Lachnospiraeae, Bacteroides, and Planococcaceae in the nasal microbiota [Noncellular mediators such as AMPs, cytokines, chemokines, immunoglobulins, arachidonic acid metabolites, and reactive oxygen species (ROS) also contribute to inflammation and thereby perturbate the UA microbiota. The nasal microbiota of subjects with increased concentrations of IL-6 is enriched with oraxella . High lecrobiota .S. aureus within their UAs had distinct levels of inflammatory biomarkers in nasal lavage samples [S. aureus in diseased UAs could further contribute to the inflammatory response [S. aureus in low-infection colony numbers triggers the secretion of the anti-inflammatory cytokine IL-10, yet when present in higher colony numbers, it reduces IL-10, leading to a more proinflammatory response [Disease states that drive UA chronic inflammation, such as cystic fibrosis (CF), obstructive sleep apnea, CRS, and asthma, also impact the UA inflammatory profile and microbiota . CF pati samples . Notable samples . This suresponse . S. aureresponse ,66,67.Acute changes in environmental factors cause perturbations to the UA microbiota ,68,69. IS. pneumoniae colonization throughout winter [Proteobacteria and increased carriage of Fusobacteria and Cyanobacteria [Haemophilus across spring and summer [Moraxella [M. catarrhalis and coronavirus is detected during the winter, and Klebsiella pneumoniae presents with higher densities in the summertime [Seasons contribute to changes in the UA microbiota . Infantst winter ,71,72. Wbacteria , whereasd summer . During oraxella . In adul2.5 and NO2) is associated with Ružička dissimilarity and the abundance of Corynebacteriaceae (NO2 only) in healthy infants [Air pollution, which is a mixture of harmful gases and particulate matter (PM), is a major contributor to dysbiosis and many respiratory diseases ,75. Glob infants .S. pneumoniae were detected in children with smoking parents [Cigarette smoke is a major contributor to disease and directly contacts the UA surfaces ,86,90,91 parents ,87,88. C parents ,82,92,93 parents ,91,94. F parents ,82,85. P parents ,82,95.Streptococcic-dominated UAs evoked proinflammatory, T-helper 1 (TH1) responses and encoded an ansamycin biosynthesis gene pathway [Pseudomonadaceae evoked a TH1 proinflammatory response and activated tryptophan metabolism gene pathways instead [CRS, defined as inflammation within the UA sinuses for more than a period of 12 weeks, is well studied in relation to the UA microbiota . In CRS  pathway . Similar instead . The dev instead .Additionally, the nasal microbiota potentially plays a vital role in regulating the local immune response, and consequently the pathophysiology of allergic rhinitis (AR) . SamplesStaphylococcus spp., while other beneficial bacteria in their microbiota decrease in abundance [Genetic disorders such as CF and primary ciliary dyskinesia (CD) also impact the UA microbiota, as they affect an individual’s mucociliary clearance ,119. Chibundance ,119. CD bundance . EquallyStreptococcus spp. is a strong predictor of future asthma development [Bacteroidetes and Proteobacteria dominated the nasal microbiota of asthma patients  whilst promoting others, such as Staphylococci [S. pneumoniae alters the carriage of S. aureus and H. influenzae, yet S. pneumoniae colonization remains unchanged [Unsurprisingly, multiple therapeutics impact the UA microbiota, such as saline rinses, intranasal corticosteroids, probiotics, and antibiotics. Treatments such as antibiotics directly impact the microbiota with their antimicrobial properties and lead to a decline in UA microbial diversity and biomass ,130. Theylococci ,130,135.nchanged . Intrananchanged . Nasal rnchanged . The inenchanged . Howevernchanged ,140,141.nchanged . The pronchanged . It inflnchanged ,143. Patnchanged ,143.Enterococcus faecalis administration in children with acute sinusitis reduces the frequency and duration of disease [Lactobacillus rhamnosus exemplifies this by increasing Th1 cells and decreasing Th2 cells in rodent models [Probiotics are a promising therapeutic to dysbiosis and are used in many diseases, such as asthma and CRS . Probiot disease ,152. Pro disease ,155,156.t models ,158. Mort models .Pseudomonas (see et al. [Most of the UA microbiota knowledge is obtained through cultivation assays and next-generation sequencing. Both short reads provide basic information about the UA microbial diversity and taxonomic composition of the UA microbiota. Overcoming said limitations requires long-read technologies such as Oxford Nanopore  or Pacifonas see , and reme et al. .Overall, the UA microbiota literature suggests that the development of a diverse microbiota across the UAs aids in maintaining healthy and disease-free UAs. Age, diet, environmental exposures, infections, and therapeutics all contribute to UA microbiota and health. The loss of UA community segregation appears to contribute to inflammation and disease that results in disadvantageous microbial–microbial and microbial–host interactions. Maintaining UA microbiota homeostasis may represent a major approach for future therapeutics. New approaches to sampling and analysing microbiota diversity will aid in our understanding of UA diseases. Future scientific advancements to recover the healthy microbiota may become a common choice in the treatment of patients with UA diseases.