Technical replicates, such as the number of sections analyzed or replicates for qRT-PCR analyses are indicated in the figure legend and the respective material and methods section, respectively. Statistical significance was assessed by an unpaired t-test using GraphPad Prism version 6.
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The authors find 1 using Wnt reporter technologies, that Wnt signaling is undetectable in CVOs throughout development in mice and in adult zebrafish. This manuscript is an interesting and valuable contribution to the field that deserves publication in eLife. The manuscript is well-written and well-illustrated in general. Please see if these are speedily do-able and if not pleased adequately discuss these points.
It will be useful to have this experimentally addressed, though. A more accurate analysis of the permeability properties of SFO vessels and of tight junction organization and more accurate analysis of astrocyte polarization and matrix protein organization are also asked. More specifically, the authors should make better images for their c-fos data and show that it is indeed neurons that express c-fos. The authors could remove the circadian Cldn5 data premature , the Aqp4 and CD13 data confusing.
It is known that endothelial cells can produce soluble neural mediators. This is a crucial aspect for a correct interpretation of the results. Would the authors be able to see the same c-fos upregulation in neural cells by limiting permeability with different tools? Src inhibitors? VEGF inhibitors? If this these experiments are not speedily addressable, they should be carefully and frontally discussed. It could be, therefore, that it is already undetectable at E This is not irrelevant to understand SFO vascularization mechanisms and function.
Here too, if experiments to address this are not speedily feasible, please discuss this point adequately. The authors need to show some staining with Occludin antibodies.
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However, astrocytes do not appear to be polarized AqP4 is expressed all over the parenchyma, rather than restricted to blood vessels. Are other astrocyte polarity markers e. Dystroglycan or Kir 4.
One potential explanation for the observation that astrocytes do not form proper endfeet in the SFO region could be due to the absence of basement membrane proteins. Underwater restrictions, mice may show abnormal behaviors under specific behavioral tasks e. Goltstein et al. It may be useful to test if there are any potential behaviors e.
The evidence for circadian regulation of Cldn5 expression weak. While the quantifications in Figure 4E may appear convincing, the immunostainings shown in Figure 4A-D are not convincing.
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The amount of vasculature captured in each section appears hugely variable and the Podxl staining for endothelium seems highly variable in strength. Podxl should be luminal and Cldn5 junctional or is it? Also, if true, the circadian variation in Cldn5 immunoreactivity raises a number of important issues that should be addressed, including if it is junctional or non-junctional Cldn5 that varies, if the effect is transcriptional or not if other tight junction proteins such as occludin and ZO-1 are also regulated, and more. If it is true that TJs are regulated in a circadian fashion, it is obviously interesting, but the physiological consequences are unclear, given that Plvap expression is maintained, and therefore presumably fenestrations not regulated.
Would TJ regulation matter for the permeability if fenestrations are kept? The authors speculate that the possible circadian regulation of Cldn5 could influence the circadian regulation of thirst and drinking behavior, but this is pure speculation, as far as I can judge. Again, how can Cldn5 matter on top of presumably open fenestrations? Another area where conclusions are weak relates to the TEM analysis Figure 6.
The images shown do not appear to support the conclusions that vessels in the GOF mice are slimmer, less tortuous and have less ECM deposition. The data regards c-fos expression by neurons in the SFO are also an area where the conclusions do seem robust. Neuronal staining to show that this is neuronal and not glial localization is not there. Also, some of the staining patterns are puzzling.
Should not c-fos should look similar at 0h and isotonic NaCl panels in Figure 7, but they look hugely different, casting doubt over the quantifications in Figure 7E and F. The authors find increased expression of Sox17 along with Cldn5 in GOF experiments and note in conjunction with the TEM data that this may signify arterial specification.
However, this is unfounded speculation in my opinion. There are many markers for arterial EC in the brain see Vanlandewijck et al.
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The authors should remove this statement or investigate possible arterialization more thoroughly. The authors investigate Aqp4 and CD13 staining patterns to check "other phenotypic BBB features", but although neither Aqp4 nor CD13 was found changed in the GOF situation, it is unclear why the experiments were done in the first place. In what way do the negative data display an absence of BBB features? Does the staining reflect lack of end-foot restricted localization in astrocytes, or is it the tanycytes that express Aqp4 in the CVOs?
Also, what does the CDstaining mean? Is this mural cells or perivascular fibroblasts see Vanlandewijck et al. We thank the reviewer for getting into this discussion. Representative immunofluorescent images A.
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Although thoroughly addressing this question in vivo is a project on its own and would be beyond the scope of this manuscript, we have made an effort to repeat the water restriction experiments, using the Wnt pathway activator BIO-X Author response image 1 as well as the Scr-inhibitor AZD Author response image 2. We systemically administered BIO-X by i. For the final 72 hours, mice were water-deprived and subsequently sacrificed and analyzed for Meca32 as well as for Cldn5. Unfortunately, we cloud not detect significant down regulation of Meca32 and up regulation of Cldn5 in SFO vessels.
Instead, the phenotype of physiologically leaky vessels persisted. Scr inhibition did not result in any effects on Meca32 and Cldn5 expression evidenced by IF staining. Analysis of c-fos showed the expected increased staining in the SFO of water-restricted mice data not shown. Additional experiments with other compounds such as VEGF-inhibitors could not be performed due to time limitation.
We thank the reviewer for this comment. We completely agree and have also analyzed earlier stages of BAT-gal reporter mice however, E Indeed, at this timepoint angiogenesis of the SFO is ongoing.
Consequently, due to the fact that E We have now introduced this explanation in the Results section. We thank the reviewer for this important comment. The physiological assessment of barrier tightness is indeed one of the most relevant parameters to address changes in permeability. The detailed procedure is incorporated in the Material and Methods section of the revised manuscript. This result is now shown in Figure 6.
Unfortunately, the two experiments with the new Lot of the tracer failed and due to the restricted availability of double transgenic mice and of time for the revision we were not able to repeat this experiment. In the pilot experiment vessels were nicely filled and labeled by 3 kDa TMR-dextran arrows. In the SFO dashed line tracer extravasation was visible.
We thank the reviewer for this question. The staining procedure and the antibody is incorporated in the Material and methods section of the revised manuscript. We now show that beside Cldn5, also Ocln localization at cell-cell junctions is significantly increased in GOF mice Figure 5 of revised manuscript. As mentioned above, this might be due to the masking of the specific endothelial RNA by the total RNA isolated from the whole mount preparations, which inevitably include the ependymal epithelium that also expresses Ocln. This is now discussed in the Discussion section.
We thank the reviewer for bringing up this interesting issue.
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Indeed, we could not observe Aqp4 polarisation at astrocytic endfeet in the control conditions, but equally not along with barrier tightening in GOF mice. Specifically, dystroglycan shows weak and not strictly polarized staining around SFO vessel, whereas Kir4. The co-localized distribution of dystroglycan and Kir4.