The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood–brain barrier formation and maturation
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Evaluation Summary:
This study advances our understanding of the Moody G-protein coupled receptor (GPCR) pathway in blood-brain barrier development, and describes a new role for protein kinase A (PKA) and two downstream signaling molecules in this process. It is not entirely surprising that PKA is involved, as it is downstream of many/most GPCRs, but the reciprocal localization and signaling relationship that the authors describe within subperineurial glia for Moody/PKA is very interesting. Generally, the data look very good, and the electron microscopy work is particularly nice. With some improved statistical analyses, this manuscript will make an interesting contribution to the field of neurodevelopment.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The blood–brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA’s molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.
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Author Response:
Reviewer #2 (Public Review):
Here the authors explore the role of PKA signaling in signaling downstream of the Moody GPCR in the BBB. The discovery that PKA is involved is interesting but not entirely surprising, as it functions downstream of many GPCRs to execute function (the really interesting question is how the same signal, changes in cAMP, causes PKA to do different things). The authors make the claim of a monotonic relationship between septate junctions (SJs) and cell-cell contact zones. I do not think they have measured the necessary parameters in a way that allows them to claim a "monotonic relationship between PKA activity, membrane overlap and the amount of SJ components in the area of cell contact." There is a correlation, but that is probably overstating it. There is an interesting analysis of several …
Author Response:
Reviewer #2 (Public Review):
Here the authors explore the role of PKA signaling in signaling downstream of the Moody GPCR in the BBB. The discovery that PKA is involved is interesting but not entirely surprising, as it functions downstream of many GPCRs to execute function (the really interesting question is how the same signal, changes in cAMP, causes PKA to do different things). The authors make the claim of a monotonic relationship between septate junctions (SJs) and cell-cell contact zones. I do not think they have measured the necessary parameters in a way that allows them to claim a "monotonic relationship between PKA activity, membrane overlap and the amount of SJ components in the area of cell contact." There is a correlation, but that is probably overstating it. There is an interesting analysis of several markers. These cells are very small and it is not clear what do the cytoskeletal markers really tell us. The markers change, no doubt, and do so in a way that correlates with the proposed Moody/PKA antagonistic relationship. The markers do change at the edges of cells and in regions of overlap, but wouldn't that be expected based on the changes in morphology? Again, the claim for "monotonic" changes is probably overstating the relationship.
We appreciate the suggestion and made the change of the word “monotonic” to “major”. Our results suggest that the primary role of Moody/PKA in this process is to regulate the membrane contact area between neighboring cells. This is consistent with the results of a temporal analysis of epithelium formation and SJ insertion in late embryos of WT and Moody pathway mutants, which shows that membrane contact precedes and is necessary for the appearance of SJs (Schwabe et al., 2017). Our ssTEM-based 3D reconstruction shows that the total area covered by SJs and the length of individual contiguous SJ segments are independent parameters. The latter appears to be critical for the paracellular seal, consistent with the idea that Moody plays a role in the formation of continuous SJ stands.
Doesn't the fact the total SJ area covered remains at 30% whether there is more or less overlap also argue against this (i.e. 30% of more overlap is not the same of 30% of less overlap…so more or less SJs are being made)?
Our ssTEM analysis of the larval SPG epithelium clarifies the relationship between the inter-cell membrane overlap and SJ organization and function at the ultrastructural level. This analysis indicates that the percentage of septate junction areas remains constant (at about 30%) across different PKA activity levels. This proportionality suggests a mechanism that couples cell contact with SJ formation. The finding that the surface area occupied by SJs did not significantly change, irrespective of the absolute area of cell contact, suggests an intrinsic, possibly steric limitation in how much junction can be fitted into a given cell contact space.
The study would need to be strengthened by more rigorous quantification. There is no quantification in figure 3. This is a primary point in the manuscript-that cytoskeletal markers change (in a claimed "monotonic" way) in subperineurial glia when PKA is altered.
We agree, and as suggested by the reviewer we now added quantifications for all cytoskeletal markers used in figure 3 (including GFPactin, TauGFP, EB1GFP, NodGFP, and two endosome markers Rab4RFP, Rab11GFP). The additional data are now presented in the right column of Figure 3. Technical details are provided in the method section.
There is also no quantification or statistics in Figure 5, which is among the most interesting observations.
The complementary localization of Moody and the PKA catalytic (activated) subunit is very nice. It shows a very interesting cellular polarity. However, it is unclear whether this is altered in Moody mutants (the authors only did knockdown) and whether catalytic (activated) PKA now goes everywhere.
In response to the reviewer’s suggestion, we further examined the subcellular distribution of Moody and Pka-C1 under gain- and loss-of-function conditions of Moody/PKA signaling in SPG and revised Fig. 5. We show lateral views of the CNS/hemolymph border for each condition, and line scans of fluorescence intensities for each channel along the apical-basal axis. Our results clearly demonstrate that the polarization of Moody and PKA depends on the activity of the Moody/PKA signaling pathway. In WT (Fig. 5A), Moody localized to the apical side and PkaC1 was enriched at the basal side of SPG. Under loss of Moody signaling (moody>MoodyRNAi) (B), PKA spread throughout the cell and lost its basal localization. Moody lost its apical localization in response to reduced (C) or increased PKA activity (E). Under GPCR gain-of-function. conditions (D), Pka-C1 was basally polarized, while Moody lost its asymmetric localization in SPG.
Throughout, the authors use some very nice genetic studies, using loss-of-function, gain-of-function, and enhancer/suppressor approaches, and their findings are consistent with polarized localization of Moody being important.
Reviewer #3 (Public Review):
Proper sealing of the blood brain barrier (BBB) is essential for viability in many animals, including humans and Drosophila. In Li et al., the authors used Drosophila as a simple genetic system to define the signaling pathways that control BBB formation and maintenance. In Drosophila, the BBB is composed of a thin epithelial sheath of subperineural glia (SPG) that are connected by septate junctions. Previously, the authors found that the G protein-coupled receptor Moody is essential for BBB formation during embryogenesis, but the downstream signaling pathways that facilitate septate junction assembly were not known. Here, they performed a series of genetic screens and epistasis experiments to uncover that Moody and PKA antagonistic signaling drives BBB assembly and expansion throughout organismal development. In the present study, they show that loss of PKA signaling components results in a leaky BBB both during development, and during adulthood. They further show that these functions of PKA are dependent on downstream suppression of Rho and changes in cytoskeletal dynamics. Interestingly, overexpression of PKA also causes BBB permeability, indicating that PKA signaling levels must be tightly regulated for BBB integrity. The authors then use serial section TEM to visualize the intact SPG sheath for the first time at ultrastructural resolution, and show that overexpression of PKA results in an enlarged yet patchy septate junction, accounting for the leakiness. In sum, the authors show that the combined signaling of Moody (apically located) and PKA (basally located) shapes the cytoskeleton to drive efficient assembly and maintenance of septate junctions, and thus, the BBB.
The conclusions of this paper are mostly well supported by the data, but the study would be improved by some expanded analyses and descriptions of statistical assessment.
We performed additional analysis and added more statistical data throughout the entire manuscript. All changes have been marked in the article.
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Reviewer #3 (Public Review):
Proper sealing of the blood brain barrier (BBB) is essential for viability in many animals, including humans and Drosophila. In Li et al., the authors used Drosophila as a simple genetic system to define the signaling pathways that control BBB formation and maintenance. In Drosophila, the BBB is composed of a thin epithelial sheath of subperineural glia (SPG) that are connected by septate junctions. Previously, the authors found that the G protein-coupled receptor Moody is essential for BBB formation during embryogenesis, but the downstream signaling pathways that facilitate septate junction assembly were not known. Here, they performed a series of genetic screens and epistasis experiments to uncover that Moody and PKA antagonistic signaling drives BBB assembly and expansion throughout organismal …
Reviewer #3 (Public Review):
Proper sealing of the blood brain barrier (BBB) is essential for viability in many animals, including humans and Drosophila. In Li et al., the authors used Drosophila as a simple genetic system to define the signaling pathways that control BBB formation and maintenance. In Drosophila, the BBB is composed of a thin epithelial sheath of subperineural glia (SPG) that are connected by septate junctions. Previously, the authors found that the G protein-coupled receptor Moody is essential for BBB formation during embryogenesis, but the downstream signaling pathways that facilitate septate junction assembly were not known. Here, they performed a series of genetic screens and epistasis experiments to uncover that Moody and PKA antagonistic signaling drives BBB assembly and expansion throughout organismal development. In the present study, they show that loss of PKA signaling components results in a leaky BBB both during development, and during adulthood. They further show that these functions of PKA are dependent on downstream suppression of Rho and changes in cytoskeletal dynamics. Interestingly, overexpression of PKA also causes BBB permeability, indicating that PKA signaling levels must be tightly regulated for BBB integrity. The authors then use serial section TEM to visualize the intact SPG sheath for the first time at ultrastructural resolution, and show that overexpression of PKA results in an enlarged yet patchy septate junction, accounting for the leakiness. In sum, the authors show that the combined signaling of Moody (apically located) and PKA (basally located) shapes the cytoskeleton to drive efficient assembly and maintenance of septate junctions, and thus, the BBB.
The conclusions of this paper are mostly well supported by the data, but the study would be improved by some expanded analyses and descriptions of statistical assessment.
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Reviewer #2 (Public Review):
Here the authors explore the role of PKA signaling in signaling downstream of the Moody GPCR in the BBB. The discovery that PKA is involved is interesting but not entirely surprising, as it functions downstream of many GPCRs to execute function (the really interesting question is how the same signal, changes in cAMP, causes PKA to do different things). The authors make the claim of a monotonic relationship between septate junctions (SJs) and cell-cell contact zones. I do not think they have measured the necessary parameters in a way that allows them to claim a "monotonic relationship between PKA activity, membrane overlap and the amount of SJ components in the area of cell contact." There is a correlation, but that is probably overstating it. There is an interesting analysis of several markers. These cells …
Reviewer #2 (Public Review):
Here the authors explore the role of PKA signaling in signaling downstream of the Moody GPCR in the BBB. The discovery that PKA is involved is interesting but not entirely surprising, as it functions downstream of many GPCRs to execute function (the really interesting question is how the same signal, changes in cAMP, causes PKA to do different things). The authors make the claim of a monotonic relationship between septate junctions (SJs) and cell-cell contact zones. I do not think they have measured the necessary parameters in a way that allows them to claim a "monotonic relationship between PKA activity, membrane overlap and the amount of SJ components in the area of cell contact." There is a correlation, but that is probably overstating it. There is an interesting analysis of several markers. These cells are very small and it is not clear what do the cytoskeletal markers really tell us. The markers change, no doubt, and do so in a way that correlates with the proposed Moody/PKA antagonistic relationship. The markers do change at the edges of cells and in regions of overlap, but wouldn't that be expected based on the changes in morphology? Again, the claim for "monotonic" changes is probably overstating the relationship. Doesn't the fact the total SJ area covered remains at 30% whether there is more or less overlap also argue against this (i.e. 30% of more overlap is not the same of 30% of less overlap...so more or less SJs are being made)?a
The study would need to be strengthened by more rigorous quantification. There is no quantification in figure 3. This is a primary point in the manuscript-that cytoskeletal markers change (in a claimed "monotonic" way) in subperineurial glia when PKA is altered. There is also no quantification or statistics in Figure 5, which is among the most interesting observations.
The complementary localization of Moody and the PKA catalytic (activated) subunit is very nice. It shows a very interesting cellular polarity. However, it is unclear whether this is altered in Moody mutants (the authors only did knockdown) and whether catalytic (activated) PKA now goes everywhere.
Throughout, the authors use some very nice genetic studies, using loss-of-function, gain-of-function, and enhancer/suppressor approaches, and their findings are consistent with polarized localization of Moody being important.
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Reviewer #1 (Public Review):
This work investigates the structure and maintenance of the blood brain barrier (BBB) in Drosophila. Previous work from this lab and others have shown that the BBB is composed of a specialized type of glia called the subperineurial glia (SPG), which enwrap the entire central nervous system (CNS). Furthermore, they previously identified the Moody G protein coupled receptor (GPCR) as being specifically expressed in SPG and required for BBB formation and maintenance.
Here they show that Moody protein is localized to the apical membrane domain (facing the CNS) while Protein Kinase A (PKA) is localized to the complementary basal membrane domain (facing the hemolymph of the body cavity). Not only do Moody and PKA have non-overlapping subcellular localization, but genetic interactions show that Moody and PKA act …
Reviewer #1 (Public Review):
This work investigates the structure and maintenance of the blood brain barrier (BBB) in Drosophila. Previous work from this lab and others have shown that the BBB is composed of a specialized type of glia called the subperineurial glia (SPG), which enwrap the entire central nervous system (CNS). Furthermore, they previously identified the Moody G protein coupled receptor (GPCR) as being specifically expressed in SPG and required for BBB formation and maintenance.
Here they show that Moody protein is localized to the apical membrane domain (facing the CNS) while Protein Kinase A (PKA) is localized to the complementary basal membrane domain (facing the hemolymph of the body cavity). Not only do Moody and PKA have non-overlapping subcellular localization, but genetic interactions show that Moody and PKA act antagonistically in BBB maintenance. They find that both too little and too much PKA activity disrupts the BBB.
The authors also generate a serial section Transmission Electron Microscopy (TEM) volume to analyze wild type, PKA hyperactivity, and PKA loss of function animals. They find that loss of BBB function is sue to gaps in the BBB, rather than thinning of the BBB. This conclusion is somewhat weak, however, because they did not analyze a genotype that has thin BBB structure but normal BBB function.
Their work raises the question of how does PKA promote BBB integrity. They analyze two potential PKA targets (myosin light chain kinase [MLCK], and Rho1), finding that reduced Moody levels lead to disrupted subcellular localization of both proteins in SPG; that reducing MLCK or Rho1 levels causes failure of BBB function; and that reducing Moody can rescue these phenotypes. They conclude that Moody acts antagonistically to PKA/MLCK/Rho1 to establish distinct apical and basal membrane domains in SPG which are required for BBB function.
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Evaluation Summary:
This study advances our understanding of the Moody G-protein coupled receptor (GPCR) pathway in blood-brain barrier development, and describes a new role for protein kinase A (PKA) and two downstream signaling molecules in this process. It is not entirely surprising that PKA is involved, as it is downstream of many/most GPCRs, but the reciprocal localization and signaling relationship that the authors describe within subperineurial glia for Moody/PKA is very interesting. Generally, the data look very good, and the electron microscopy work is particularly nice. With some improved statistical analyses, this manuscript will make an interesting contribution to the field of neurodevelopment.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
This study advances our understanding of the Moody G-protein coupled receptor (GPCR) pathway in blood-brain barrier development, and describes a new role for protein kinase A (PKA) and two downstream signaling molecules in this process. It is not entirely surprising that PKA is involved, as it is downstream of many/most GPCRs, but the reciprocal localization and signaling relationship that the authors describe within subperineurial glia for Moody/PKA is very interesting. Generally, the data look very good, and the electron microscopy work is particularly nice. With some improved statistical analyses, this manuscript will make an interesting contribution to the field of neurodevelopment.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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