Glypicans specifically regulate Hedgehog signaling through their interaction with Ihog in cytonemes
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Abstract
The conserved family of Hedgehog (Hh) signaling proteins plays a key role in cell-cell communication in development, tissue repair and cancer progression. These proteins can act as morphogens, inducing responses dependent on the ligand concentration in target cells located at a distance. Hh proteins are lipid modified and thereby have high affinity for membranes, which hinders the understanding of their spreading across tissues. Direct contact between cell membranes by filopodia-like structures (also known as cytonemes) could be the simplest explanation for Hh dispersal. To better understand this signaling mechanism, we have analyzed in Drosophila the interaction between the glypicans that, besides for other pathways, are necessary for Hh signaling, plus the adhesion molecules and Hh coreceptors Ihog and Boi. We describe that glypicans (Dally and Dally-like protein) are required to maintain Ihog, but not Boi, protein levels. We also show that ectopic Ihog stabilizes cytonemes through its interaction with glypicans, and we determine that two Ihog fibronectin III domains are essential for this interaction. Our data suggest that this interaction with Ihog in cytonemes confers the specificity of glypicans for Hh signaling.
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Reviewer #3:
The glypicans Dally and Dlp have important roles in morphogen signaling, and this work is of particular interest for me because it significantly advances our understanding of the multiple roles they appear to have in signal processing, signal presentation and signal reception. It is unfortunate that most of the literature has presented results and phenotypes in simplistic or simple-minded ways that do not recognize the different roles or the glypicans, or do not take experimental approaches that might distinguish them. This work of the Guerrero lab is an exception, as it is an important contribution to understanding these different roles, especially given the additional complexity introduced by the role of cytonemes. If its thoroughness and in-depth analysis are typical of work from this lab, so is the challenging …
Reviewer #3:
The glypicans Dally and Dlp have important roles in morphogen signaling, and this work is of particular interest for me because it significantly advances our understanding of the multiple roles they appear to have in signal processing, signal presentation and signal reception. It is unfortunate that most of the literature has presented results and phenotypes in simplistic or simple-minded ways that do not recognize the different roles or the glypicans, or do not take experimental approaches that might distinguish them. This work of the Guerrero lab is an exception, as it is an important contribution to understanding these different roles, especially given the additional complexity introduced by the role of cytonemes. If its thoroughness and in-depth analysis are typical of work from this lab, so is the challenging presentation that makes understanding it so difficult. My recommendation to the authors is to clearly describe the different roles that have been attributed to the glypicans and for every experiment they present, clearly articulate how the results might implicate or distinguish any or several of them.
Although the figures are excellent, the manuscript is not well-written and would benefit from a rewrite.
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Reviewer #2:
This manuscript interrogates function of Ihog and Boi adhesion molecules in cytoneme-based transport of the Hedgehog morphogen in Drosophila. The cell biology of how cytonemes are regulated to deliver morphogen signals is not yet well understood, so the work addresses an important topic that will be of interest to a broad audience. However, much of the study refines previous work from the same group to provide only a modest advance in understanding of how Ihog impacts cytoneme behavior.
The authors use genetic strategies in Drosophila to investigate how Ihog and Boi influence cytoneme dynamics. They find that the two proteins act differently with regard to cytoneme function. Boi effects are not exhaustively analyzed, but a number of genetic experiments are performed to interrogate Ihog. The authors reveal that the …
Reviewer #2:
This manuscript interrogates function of Ihog and Boi adhesion molecules in cytoneme-based transport of the Hedgehog morphogen in Drosophila. The cell biology of how cytonemes are regulated to deliver morphogen signals is not yet well understood, so the work addresses an important topic that will be of interest to a broad audience. However, much of the study refines previous work from the same group to provide only a modest advance in understanding of how Ihog impacts cytoneme behavior.
The authors use genetic strategies in Drosophila to investigate how Ihog and Boi influence cytoneme dynamics. They find that the two proteins act differently with regard to cytoneme function. Boi effects are not exhaustively analyzed, but a number of genetic experiments are performed to interrogate Ihog. The authors reveal that the extracellular domains of Ihog interact with the glypicans Dally and Dlp to stabilize cytonemes that originate from Ihog over-expressing cells. Knockdown of Ihog does not alter cytoneme dynamics.
The most novel aspect of the study - that Boi functions differently than Ihog in cytonemes - is, unfortunately, not expanded upon. Some experiments lack controls or are presented in a manner that prevents clear interpretation of results.
Key points to be addressed:
Figure 1: Null alleles and RNAi silencing are used interchangeably to reduce Ihog, Boi, Dally and Dlp function in vivo. Results between methods are directly compared. Oftentimes, controls are not included to confirm the level of knockdown following RNAi. If possible use null alleles due to consistency. However, if this is not possible due to experimental reasons, give an explanation and state impact in the discussion.
Ihog levels decrease following loss of Dally or Dlp and Boi levels appear to increase following knockdown of Ihog, Dally, or Dlp. These stability changes have previously been reported. The mechanism is not clear, so should have been investigated here - especially the increased Boi protein level. How does this occur? Is stabilization occurring at the protein level or is gene expression changing? Is this a compensatory upregulation?
Based upon the supplement for Figure 2, it looks like the Ihog truncation mutants show variable stability. Might this be affecting the extent to which they alter Dally or Dlp stability? The western blot data are presented as crops of single bands adjacent to crops of a molecular weight ladder. Blots should be shown as intact images, preferable with all variants compared across a single gel with a loading control. As presented, relative stability/expression levels are impossible to assess.
Figures 3-4: Ihog mutant transgenes are tagged with either HA or RFP. Best to be consistent with tags when mutant function is being directly compared. Given that the HA tag is a small epitope and the RFP is a protein tag, they may differentially alter protein functionality. To be consistent it would be preferable to use the same tags. However, if this is not possible due to experimental reasons, the technical implication can also be mentioned in the discussion.
Figure 5: Investigation of histoblast cytonemes reaching into ttv, botv mutant clones: The ability of cytonemes to invade double mutant clones is altered only under the engineered situation of glypican dysfunction combined with Ihog over-expression. From this, it is concluded that Ihog is acting with glypicans to stabilize cytonemes. This may be the case, but they ability to see it only under an engineered situation of compound mutation plus Ihog over-expression leads this review to question the physiological relevance of the observation. Of similar concern is that the authors state the ability of Ihog over-expressing cell cytonemes to cross small vs. large ttv, botv clones differs. The difference is very difficult to appreciate from the results presented.
Figure 6: The apparent functional difference between Ihog and Boi in the ability to stabilize cytonemes is potentially very interesting, but is not investigated, which limits the advance of the current study.
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Reviewer #1:
In the article "Glypicans specifically regulate Hedgehog signalling through their interaction with Ihog in cytonemes" Simon et al. elucidate the function of Glypicans in Hh transport via cytonemes. The manuscript describes convincingly that the fly glypicans Dally and Dally-like are required to maintain the expression of the Hh co-receptor Ihog. Ihog - in turns - stabilises Hh cytonemes through its interaction with Glypicans to establish the Hh gradient in the wing imaginal disc. The authors further carried out an extensive molecular analysis of Ihog and identified the relevant domains within the protein required for interactions with Glypicans, Patched, and Hh. In general, this is a very thorough, detailed analysis of Ihog function. The images and videos are excellent. However, prior publication, there are two major …
Reviewer #1:
In the article "Glypicans specifically regulate Hedgehog signalling through their interaction with Ihog in cytonemes" Simon et al. elucidate the function of Glypicans in Hh transport via cytonemes. The manuscript describes convincingly that the fly glypicans Dally and Dally-like are required to maintain the expression of the Hh co-receptor Ihog. Ihog - in turns - stabilises Hh cytonemes through its interaction with Glypicans to establish the Hh gradient in the wing imaginal disc. The authors further carried out an extensive molecular analysis of Ihog and identified the relevant domains within the protein required for interactions with Glypicans, Patched, and Hh. In general, this is a very thorough, detailed analysis of Ihog function. The images and videos are excellent. However, prior publication, there are two major criticisms, which needs to be addressed, in my opinion.
Firstly, the first part of the manuscript, the molecular analysis of Ihog (Fig.1-4) seems to be detached from the second cytoneme-focussed part (Fig. 5, 6). Independent evidence is needed to show support for the idea that the Ihog-Gly mediated stabilisation of cytonemes is responsible for the expansion of the signalling gradient. Are the static cytonemes involved in a flattened gradient or are the receiving cells just sensitised for Hh? Can cytonemes be (de-) stabilised w/o interfering with Hh components to untangle these observations? The authors write "Intriguingly, the same Ihog domains that regulate cytoneme dynamics are those also involved in the recruitment of Hh ligand, glypicans and the reception complex."
My concern is that cytoneme dynamics and Hh gradient formation could be two parallel, independent events -> one needs to show this interdependency in a clear way. I could imagine an analysis of the consequences when Ihog is overexpressed, and cytoneme formation is inhibited (by other means). Consistently, could one stabilise cytonemes in an Ihog-reduced background and analyse gradient formation?
Secondly, the authors demonstrate an effect of Ihog alterations on the formation of the gradient. However, what is the physiological relevance? What are the consequences of Ihog/Gly-mediated cytoneme stabilisation and gradient formation on tissue patterning and wing formation? If this is not possible to show experimentally, this needs to be discussed.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 12 2021, follows.
Summary
In summary, this manuscript elucidates the function of Glypicans in Hh transport via cytonemes. The reviewers felt that that the manuscript describes convincingly that the fly glypicans Dally and Dally-like are required to maintain the expression of the Hh co-receptor Ihog, which stabilises cytonemes to establish the Hh gradient in the wing imaginal disc. A molecular analysis of Ihog domains was well executed.
Although the manuscript provides an in-depth analysis, the reviewers believe that the presentation of the data is rather challenging for the readers. The authors need to clearly describe the different roles that have been attributed to the glypicans and for every experiment presented, a clear …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 12 2021, follows.
Summary
In summary, this manuscript elucidates the function of Glypicans in Hh transport via cytonemes. The reviewers felt that that the manuscript describes convincingly that the fly glypicans Dally and Dally-like are required to maintain the expression of the Hh co-receptor Ihog, which stabilises cytonemes to establish the Hh gradient in the wing imaginal disc. A molecular analysis of Ihog domains was well executed.
Although the manuscript provides an in-depth analysis, the reviewers believe that the presentation of the data is rather challenging for the readers. The authors need to clearly describe the different roles that have been attributed to the glypicans and for every experiment presented, a clear explanation of the impact of the results is needed e.g. Figure 5. In addition, the stability of Ihog and Boi by altered Glypican levels and their ability to stabilize cytonemes needs to be investigated. Finally, linking the Ihog analysis to cytoneme stability analysis needs improvement.
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