Infections and diseases in the brain and spine can be very damaging and debilitating. Indeed, the central nervous system also needs a carefully controlled biochemical environment to survive. As such, all animals with a backbone have barriers and defenses to protect and preserve this key system. One of these is the blood-brain barrier, a physical barrier between the brain and the outside world. Where most blood vessels allow relatively free exchange of chemicals between the blood and surrounding cells, the blood-brain barrier controls what can move between the bloodstream and the brain.

Yet, there are gaps in the blood-brain barrier, specifically within structures in the brain called the circumventricular organs. These leaky vessels allow the brain cells in these regions to monitor the blood and respond to changes, for example, by triggering sensations such as hunger, thirst or nausea. It is not clear what stops the blood-brain barrier from forming in these regions and what effect the presence of a barrier would have on the brains activity, or the health and behavior of the animal.

Benz et al. have now used mice and zebrafish to examine the development and structure of the blood-brain barrier. The investigation revealed that the signals that induce the blood-brain barrier throughout the brain are absent in the circumventricular organs of both species. Next, by artificially activating a protein involved in cell-cell interactions in mice, Benz et al. created blood-brain barrier-like structures in circumventricular organs by converting the leaky vessels into tight ones. This change meant that the brain cells in these regions did not respond properly to water deprivation, which potentially may have affected the regulation of thirst in these mice.

Understanding the blood-brain barrier could have a variety of impacts on how we treat diseases in the central nervous system. This includes stroke, brain tumors and Alzheimers disease. These findings could particularly help scientists to better understand conditions that affect basic needs like thirst and hunger.

In vertebrates, the endothelial blood-brain barrier (BBB) is crucial for providing a permissive microenvironment for neuronal function. During developmental brain vascularization, blood vessels undergo Wnt/β-catenin signaling, driven by Wnt7a/7b that is required for angiogenesis as well as for BBB formation (Daneman et al., 2009; Stenman et al., 2008; Liebner et al., 2008). In the adult, the Wnt pathway remains instrumental to maintain BBB function in endothelial cells (ECs) of the central nervous system (CNS) (Zhou et al., 2014). Herein activation of β-catenin/TCF signaling can be induced by two flavors of the canonical Wnt pathway mediated by the ligands Wnt7a/7b and the non-Wnt-related norrin disease protein (Ndp), binding to the receptor complexes frizzled-4/Lrp5/6/Gpr124/Reck and frizzled-4/Lrp5/6/Tspan12, respectively (Junge et al., 2009; Chang et al., 2017; Cullen et al., 2011; Posokhova et al., 2015; Kuhnert et al., 2010; Wang et al., 2014; Cho et al., 2017; Vanhollebeke et al., 2015; Eubelen et al., 2018).

Although a strict control of the exchange between blood and the CNS tissue by the endothelial BBB is realized in most parts of the CNS, some areas of the brain and the ciliary body of the eye are exceptions to this rule, providing a physiologically highly relevant door to the CNS. The circumventricular organs (CVOs) are a number of small midline structures found in all vertebrate brains, located around the third and fourth ventricle. CVOs have a rich capillary plexus, which physiologically lacks BBB properties (Ganong, 2000; Ufnal and Skrzypecki, 2014; Langlet et al., 2013; Benarroch, 2011). These characteristics are regarded as important sites of communication between brain and blood. Based on their function, CVOs are commonly classified into secretory and sensory organs. The median eminence, the neurohypophysis (posterior pituitary, PP), the pineal gland (PI) and the subcommissural organ (SCO) belong to the secretory group. The vascular organ of the lamina terminalis (organum vasculosum of the lamina terminalis, OVLT), the subfornical organ (SFO) and the area postrema (AP) are considered as sensory organs (Ufnal and Skrzypecki, 2014).

The leaky vessels of the CVOs evidently have different morphological and structural characteristics from those of typical BBB vessels, lacking a cellular organization as neuro-vascular unit (NVU), without distinctive astrocytic endfeet and ECs with numerous fenestrations and vesicles (Morita et al., 2016). Interestingly, in previous reports neither the tight junction proteins claudin-5 (Cldn5), occludin (Ocln) and zonula occludens 1 (Tjp1/ZO-1) (Mullier et al., 2010; Maolood and Meister, 2010; Norsted et al., 2008; Sisó et al., 2010), nor the transporter proteins glucose transporter 1 (Glut-1) and transferrin receptor (Norsted et al., 2008; Maolood and Meister, 2009), showed a BBB-like staining in leaky CVO ECs. In line with these vascular features, Morita et al. showed that 10 kDa dextran accumulates in the perivascular space between the inner and outer basement membranes, whereas smaller tracers up to 3 kDa dextran diffuse into the parenchyma (Morita et al., 2016). Not only the endothelium, but also the perivascular space in CVOs has specific properties, being enlarged and filled by collagen fibers, fibroblasts, astrocytic processes and axons (Morita and Miyata, 2012).

Beside the leaky vasculature of the CVOs, free diffusion of substances into the brain parenchyma is prohibited by tanycytes, specialized cells of the ependymal lining. Tanycytes contain long processes, unlike typical ependymal cells, which project to the parenchyma of the CVOs making contact to the fenestrated vascular wall of the CVOs (Langlet et al., 2013; Benarroch, 2011; Mullier et al., 2010).

The detailed function of all CVOs has not intensely been explored in the past, but more recently, the SFO together with the OVLT, the median preoptic nucleus (MnPO) and the PP were identified as a functional circuit, regulating drinking behavior and water homeostasis of the organism (Zimmerman et al., 2016; Oka et al., 2015; Augustine et al., 2018). The SFO is a tiny organ located underneath the fornix at the foramen of Monro, protruding into the third ventricle at the meeting point with the lateral ventricles. The dense vascular network in the SFO is similarly organized in different vertebrate species. It presents with heterogeneous vessel phenotypes and can therefore be divided into two zones. Whereas the outer shell contains more BBB expressing vessels, the majority within the ventromedial core is fenestrated with a wide perivascular space. In general, the vascular density is four to five times higher than in other brain regions with tortuous vessels, exhibiting a high blood volume and slow perfusion rate, thereby contributing to high permeability rates (Sisó et al., 2010; Duvernoy and Risold, 2007; Fry et al., 2007; Bouchaud et al., 1989).

Although a cell type-specific expression analysis by single cell sequencing, as it has been performed for the brain parenchyma (Vanlandewijck et al., 2018), has not been conducted yet, neurons and astrocytes of sensory CVOs (SFO, OVLT and AP) were shown to express specific receptors and ion channels. Those permit them to detect several blood–derived molecules such as salts, hormones, lipids and toxins and convey this information to other parts of the brain, involved in controlling autonomic and peripheral functions (Benarroch, 2011; Sisó et al., 2010). Evidence supports that 25–60% of CVO neurons respond to signals in the circulatory system and a single neuron may respond to multiple signals such as osmolarity and angiotensin II. Most sensory CVOs play a role in the control of blood pressure, fluid and sodium balance, cardiovascular regulation, feeding and energy homeostasis and immunomodulation (Ufnal and Skrzypecki, 2014; Benarroch, 2011; Sisó et al., 2010; Morita and Miyata, 2012; Smith and Ferguson, 2012).

Given the fact that endothelial Wnt/β-catenin signaling is necessary for BBB development and maintenance, the question remains if the pathway is instrumental in the establishment of vascular heterogeneity in the CNS. Hence, we addressed the question if Wnt/β-catenin signaling is operational in CVO vessels during development and if local regulation of β-catenin signaling is involved in establishing a leaky vascular phenotype in CVOs. Finally, we asked if dominant activation of β-catenin in ECs can overwrite the leaky vessel fate thereby affecting CVO function.

Here we show by investigating CVOs during BAT-gal reporter mouse development that at any embryonic stage analyzed, starting from E13.5, when the first SFO primordium could be identified, to P21, no activation of β-catenin signaling in CVO vessels could be detected. Focusing on the SFO as a crucial CVO in the regulation of water homeostasis, we show that blood vessels in the caudal portion were mainly leaky evidenced by Plvap/Meca32 staining, whereas capillaries in the rostral portion of the organ were tighter. Interestingly, within the vessel continuity, individual ECs might be Plvap/Meca32⁺//Cldn5^- followed by a Plvap/Meca32^-//Cldn5⁺ EC, suggesting a locally confined regulation of barrier properties. Dominant, genetic activation of β-catenin signaling (gain-of-function, GOF) in ECs resulted in tightening of CVO blood vessels, evidenced by the switch from a Plvap/Meca32⁺//Cldn5^- to a Plvap/Meca32^-//Cldn5⁺ vascular phenotype. Vessel tightening was accompanied by a significant reduction in endothelial fenestrations, that likely contributed to the reduction of transcellular permeability evidenced by decreased tracer leakage. Interestingly, endothelial tightening did not coincide with the formation of astrocytic endfeet towards a BBB-like NVU. Finally, we observed augmented neuronal activity in the SFO under thirst conditions after sealing CVO vessels, supported by significantly increased neuronal c-fos staining in the SFO of GOF mice.

The present study deals with the regulation of the leaky vascular phenotype in the CVOs and in the SFO in particular. Specifically, we addressed the questions, a) if the Wnt/β-catenin pathway is operational in ECs of CVOs during murine development and in the adult mouse and zebrafish, b) if endothelial-specific, dominant activation of β-catenin transcription could convert the leaky vascular phenotype in CVOs and c) if the latter may have an effect on CVO function.

The principle findings of this study are: 1) Wnt/β-catenin signaling is undetectable in CVO vessels during BAT-gal reporter mouse development; 2) similarly, β-catenin-mediated transcription is strongly reduced in the adult zebrafish OVLT; 3) SFO vessels are heterogenous regarding the expression of Plvap/Meca32 and Cldn5; 4) upon genetic β-catenin GOF in ECs, leaky SFO vessels are partially converted into tight vessels; 5) functional conversion of SFO vessel towards a BBB-like identity affects neuronal activity in the SFO.

Wnt/β-catenin is crucial for brain vascularization and BBB development, by regulating endothelial sprouting as well as by promoting a BBB expression profile in ECs, respectively (Vanhollebeke et al., 2015; Liebner et al., 2008; Daneman et al., 2009; Stenman et al., 2008; Zhou et al., 2014). CVOs are well known, but poorly investigated, structures in the midline of vertebrate brains, conferring neurosensory and/or neurosecretory function. Because of this physiological function, CVO blood vessels were described for a long time to lack BBB characteristics, a feature that is considered to be important for allowing neurons to ‘sense’ salts, hormones, lipids and toxic compounds in the blood (Sisó et al., 2010; Kiecker, 2018). Indeed, it has been shown that neurons send axons into the extended perivascular space, which is in line with their sensory function (Morita and Miyata, 2012). The peculiar, leaky specialization of the CVO vascular system is well documented, showing tortuous and fenestrated vessels with poorly developed inter-endothelial junctions (McKinley et al., 2003). However, how this specialization is induced on a molecular level during development and how it is maintained is currently not well understood. Vascular endothelial growth factor (VEGF) is the best described inducing factor for endothelial fenestrations and is reported to be expressed in sensory CVOs as well as in other tissues that physiologically require endothelial fenestrations, such as the choroid and the ciliary body of the eye (Furube et al., 2014; Ford et al., 2012; Kinnunen and Ylä-Herttuala, 2012).

As the Wnt/β-catenin pathway is considered a master switch for barriergenesis, we hypothesized that β-catenin transcription is not operational during CVO vascularization. The data provided here support this interpretation, as in BAT-gal reporter mice, from the initial identification of the SFO primordium at E13.5, none of the investigated developmental stages revealed a single β-galactosidase-positive vessel within the CVOs (Figure 1; Figure 1—figure supplements 1 and 2). Although this finding may formally not exclude low level activation of the pathway in ECs, the observation that neighboring, non-endothelial cells in the CVOs, do show Wnt pathway activation, supports the interpretation of low or absent Wnt/β-catenin signaling in CVO ECs. Specifically, we observed that the ependymal cells covering the SFO as well as stromal cells in the core of the organ show Wnt/β-catenin pathway activation (Figure 1). Quantitative RT-PCR revealed also expression of Wnt3a, Wnt7 as well as Fzd4 in the SFO, suggesting that at least the BBB-inducing machinery is expressed (data not shown). As also in the adult, β-catenin-mediated transcription in ECs is required to maintain BBB function, the absence of reporter activity in mouse (data not shown) or zebrafish models presented in this study, further underlines that Wnt is evidently not operational in CVO vessels. This may support the hypothesis that Wnt/β-catenin signaling is actively suppressed in the SFO and likely also in other CVOs that have fenestrated vessels. So far, no conclusive data are available demonstrating Wnt pathway inhibitors in the CVOs, however, it has been shown that expression of the soluble frizzled receptor protein 1 (Sfrp1) is about 30 times higher in the rat choroid plexus (CP), that also lacks BBB vessels, compared to the striatum and parietal cortex (Bowyer et al., 2013). Nevertheless, a detailed analysis of the CVOs regarding cell type-specific expression profiles has not been published yet.

Still, it has to be noted that vessels in the SFO, OVLT and PP are heterogenous regarding the expression of Plvap/Meca32 and Cldn5 (Figure 3, Figure 3—figure supplement 1). Similarly, differentially tight vessels were also shown in other CVOs (Morita and Miyata, 2012). This raises the question if in the CVOs, unlike in the brain parenchyma a ‘…gradual phenotypic change (zonation) along the arteriovenous axis…’ is realized (Vanlandewijck et al., 2018), or if alternating endothelial differentiation might be established by factors yet to be discovered.

The present findings may suggest that, at least to some degree, vascular phenotypes in the SFO are locally regulated, which would be in line with their role providing local access for neurons to the blood milieu. If vessel differentiation might also be dynamically regulated to control water homeostasis in a circadian rhythm (Gizowski et al., 2016), is currently unknown and subject to ongoing investigation. In this regard it is interesting to note however, that Cldn5 and Ocln mRNA were not significantly upregulated when analyzed in whole mount dissected SFOs from GOF mice (Figures 4H and 5D). This might be due to several reasons, such as signal masking by other vessels in the whole mount preparations. Alternatively, this finding might support the interpretation that Cldn5 and Ocln are not transcriptionally regulated by β-catenin, but rather regulated on a post-transcriptional level. Interestingly, there is still some controversy about Cldn5 regulation by Wnt/β-catenin, as it has been shown by Taddei et al. that β-catenin cooperates with FOXO1 to suppress Cldn5 at the promotor level under pro-angiogenic conditions (Taddei et al., 2008). On the other hand, it has been shown that Sox18, a member of the SOX family of high-mobility group box transcription factors, is instrumental in activating Cldn5 transcription, contributing to endothelial barrier formation (Fontijn et al., 2008). Given the high redundancy of SoxF genes (Sox7, 17, 18) (Zhou et al., 2015), it might be feasible that Sox17, that we report here to be upregulated in SFO vessels of β-catenin GOF mice (Figure 5—figure supplement 1), mediates Cldn5 regulation.

Although the regulation of Cldn5 on the promotor level and the role of β-catenin herein requires additional investigation, the tightening of SFO vessels by Cldn5 protein upregulation in β-catenin GOF mice is consistent with previous reports in other regions of the brain (Zhou et al., 2014). Interestingly, in the SFO of GOF mice, we also observed a significantly augmented junctional localization of Ocln, which is in line with the endothelial tightening, but, like for Cldn5, at which molecular level the Ocln regulation occurs remains to be clarified. Moreover, the adherens and tight junction-associated protein ZO-1 qualitatively showed a slight increase in junctional continuity in the GOF condition, which fits with the overall formation of more elaborate junctional complexes between ECs. The fact that ZO-1 exhibits also junctional staining in the controls (Figure 5—figure supplement 1), is consistent with its role in VE-cadherin-based adherens junctions, which are also formed by SFO vessels (Figure 4—figure supplement 1) (Tornavaca et al., 2015).

Beside the mere upregulation of Cldn5 and Ocln, endothelial β-catenin GOF resulted in the abolishment of fenestrations and strengthened inter-endothelial junctions. These findings are well in line with a reduction in VEGF signaling in glioma ECs upon Wnt/β-catenin activation via the downregulation of VEGF receptor 2 (VEGFR2, flk-1) and upregulation of VEGFR1 (Reis et al., 2012). This suggests that also upon β-catenin GOF in CVO vessels the responsiveness of ECs for VEGF could be reduced, leading to regression of fenestrations. Interestingly, the structural components of the NVU such as astrocytic endfeet and ECM, additional crucial BBB features, were not observed to be changed by the GOF condition (Figure 7—figure supplements 1 and 2). Also, the vessel coverage by pericytes showed no major changes in the SFO comparing GOF and controls (data not shown). If the perivascular fibroblasts, recently described by Vanlandewijck et al. (Vanlandewijck et al., 2018), are present at SFO vessels and if yes, whether they are affected by β-catenin GOF in ECs has to be determined in future investigations. Hence, to form the NVU structure might require additional cues and/or prolonged time to form, although the latter explanation might not be as likely as the first, given that even after sixty days after TAM injection the control-like phenotype persisted (data not shown). These findings support the conclusion that tightening the ECs in the SFO via β-catenin GOF does not lead to pronounced structural alterations at the NVU. As the leaky vessels of the SFO core are surrounded by a prominent perivascular space, which is considered to be important for the communication of neuronal axons with the blood milieu, it might be therapeutically beneficial that the NVU is not affected by the dominant activation of endothelial β-catenin.

One of the main questions investigated in the present work is how the vasculature in the sensory CVOs like the SFO functionally cooperates with the neurons and other stromal cells to achieve proper physiological regulation of fundamental body parameters like water homeostasis. So far, the vasculature has drawn little attention in this respect, even though considerable progress has recently been made to unravel the regulation of drinking behavior by the SFO, OVLT and the PP (Gizowski et al., 2016; Matsuda et al., 2017; Oka et al., 2015; Zimmerman et al., 2016; Augustine et al., 2018). Specifically, it was shown for the SFO that two distinct populations of neurons expressing ETV-1 and Vgat mediate thirst-ON and thirst-OFF signals, respectively (Oka et al., 2015). Here we provide evidence for an essential role of endothelial barrier function in neuronal activation in water restricted mice, as neuronal c-fos reactivity was increased in water-deprived GOF animals (Figure 9). How this finding relates to the drinking behavior and to the activity of excitatory and inhibitory neuronal signals to and from the median preoptic nucleus (MnPO), which was shown to host the behavioral output neurons (Augustine et al., 2018), is beyond the scope of this study and is subject to future work. Moreover, it remains to be clarified if the increased c-fos signal in the SFO of GOF mice is directly caused by the tightened vessel phenotype, or indirectly affected by an altered angiocrine profile of the tightened endothelium, potentially leading to altered drinking behavior. Preliminary analysis of primary mouse brain microvascular ECs (MBMECs) treated with Wnt3a revealed no regulation of VEGF that was previously described to be neuroactive (data not shown) (Mackenzie and Ruhrberg, 2012).

Although own preliminary experiments aiming to pharmacologically tighten CVO vessels with a systemically administered Wnt/β-catenin activator did not result in SFO vessel tightening (data not shown), this might be a potential way to therapeutically modulate water intake. Interestingly, patients that chronically receive LiCl, an FDA-approved drug for bipolar disorders and a potent Wnt/β-catenin activator, frequently develop polyuria that is linked to altered anti-diuretic hormone (ADH; vasopressin) function, which is released by the PP. Moreover, many patients develop polydipsia and urinate more frequently (Malhi, 2015). Hence in-depth investigation of the pharmacologic modulation of SFO vessel permeability is required. Although the detailed mechanisms underlying the neuro-vascular coupling in the CVOs have to be investigated in more detail, in light of the present work however, the CVO vasculature likely participates actively in controlling water homeostasis.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files are provided for Figures 4F-H, 5C, 6B, 7D, 8E-F, 9C as well as in Figure 4-figure supplement 1C, Figure 4-figure supplement 3D, Figure 8-figure supplement 1B. Raw data for all quantifications are provided in a separated MS Excel documents.

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Author details

1. Fabienne Benz

   Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, Drafting the article or revising it critically for important intellectual content, Final approval of the version to be published

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1891-7680

2. Viraya Wichitnaowarat

   Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   Investigation, Visualization, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

   Competing interests

   No competing interests declared

3. Martin Lehmann

   Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   Investigation, Visualization, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

   Competing interests

   No competing interests declared

4. Raoul FV Germano

   Laboratory of Neurovascular Signaling, Department of Molecular Biology, ULB Neuroscience Institute, Université libre de Bruxelles, Bruxelles, Belgium

   Contribution

   Investigation, Visualization, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1247-0689

5. Diana Mihova

   Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   Investigation, Visualization, Substantial contributions to acquisition and analysis of data, Final approval of the version to be published

   Competing interests

   No competing interests declared

6. Jadranka Macas

   Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   Visualization, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1661-2525

7. Ralf H Adams

   Department of Tissue Morphogenesis, Max-Planck-Institute for Molecular Biomedicine, University of Münster, Faculty of Medicine, Münster, Germany

   Contribution

   Resources, Funding acquisition, Final approval of the version to be published

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3031-7677

8. M Mark Taketo

   Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan

   Contribution

   Resources, Final approval of the version to be published

   Competing interests

   No competing interests declared

9. Karl-Heinz Plate

   1. Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany
   2. Excellence Cluster Cardio-Pulmonary systems (ECCPS), Partner site Frankfurt, Frankfurt, Germany
   3. German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt, Germany
   4. German Center for Cardiovascular Research (DZHK), Partner site Frankfurt/Mainz, Frankfurt, Germany
   5. German Cancer Research Center (DKFZ), Heidelberg, Germany

   Contribution

   Conceptualization, Supervision, Investigation, Methodology, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

   Competing interests

   No competing interests declared

10. Sylvaine Guérit

    Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany

    Contribution

    Resources, Funding acquisition, Final approval of the version to be published

    Competing interests

    No competing interests declared

11. Benoit Vanhollebeke

    1. Laboratory of Neurovascular Signaling, Department of Molecular Biology, ULB Neuroscience Institute, Université libre de Bruxelles, Bruxelles, Belgium
    2. Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Wallonia, Belgium

    Contribution

    Investigation, Methodology, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-0353-365X

12. Stefan Liebner

    1. Institute of Neurology (Edinger Institute), University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany
    2. Excellence Cluster Cardio-Pulmonary systems (ECCPS), Partner site Frankfurt, Frankfurt, Germany
    3. German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt, Germany

    Contribution

    Conceptualization, Data curation, Software, Supervision, Funding acquisition, Visualization, Writing—original draft, Project administration, Writing—review and editing, Substantial contributions to acquisition, analysis and interpretation of data, Final approval of the version to be published

    For correspondence

    stefan.liebner@kgu.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4656-2258

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