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Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc

Ioannis Nellas, K. Venkatesan Iyer, Juan M. Iglesias-Artola, André Nadler, Natalie A. Dye, Suzanne Eaton

Preprint posted on September 12, 2021 https://www.biorxiv.org/content/10.1101/2021.09.11.459911v1

Hedgehog and glycolysis: the perfect team to regulate energy production in the developing Drosophila wing disc.

Selected by Julia Grzymkowski

Background

Developmental tissue growth is an energy-dependent process that relies both on ATP levels and the production of biosynthetic precursors. The concept of “aerobic metabolism” (i.e., Warburg effect), where increased glucose uptake occurs despite active respiration, has been widely studied in the context of cancer. The increased glucose uptake allows cancer cells to redirect glycolytic metabolites to biosynthetic pathways needed for growth. Recently, studies have emerged showing that some developing tissues and/or cells display aerobic metabolism as well (1-3), and that glycolysis regulates developmental processes such as migration and differentiation. In addition, connections between morphogen signaling (e.g., Wnt, FGF, Hh) and metabolism were established lately in different developmental contexts, and it is of continued interest to determine how manipulating one may affect the other (1, 4). However, research in this field is in its infancy. In their preprint, Nellas et al use a fluorescent biosensor to study the spatial dynamics of ATP within the Drosophila wing disc and how Hedgehog (Hh) signaling influences energy production during development of this tissue.

Key Findings

Using a FRET-based ATP sensor, the authors found that ATP levels remain steady throughout the wing pouch, but decrease significantly upon oxidative phosphorylation (OxPhos) inhibition with antimycin A. Upon closer inspection, the authors noticed that with antimycin A treatment, ATP levels declined significantly slower in two specific regions of the wing disc as compared to the rest. These regions of slower ATP decline were within known growth “organizer” regions: in the Dorsal-Ventral (DV) boundary, which has high Wingless/Notch signaling, and near the Anterior-Posterior (AP) boundary, which has high Hh signaling. The authors posited that these organizer regions may produce more ATP through glycolysis during OxPhos inhibition, and that this may explain why ATP levels decline more slowly. Inhibiting both OxPhos and glycolysis concomitantly resulted in a steep decline in ATP levels throughout the entire wing disc, including the organizer regions. Therefore, the authors concluded that most ATP in the wing disc is generated by OxPhos, but when OxPhos is inhibited, glycolysis continues to generate significant levels of ATP within the organizer regions.

These results suggest that morphogen signaling may control energy production within these growth organizer regions. Hh is expressed in the posterior compartment of the wing disc, where it then travels to the anterior to be received by the membrane protein, Patched (Ptc). Ptc is normally a Hh signaling repressor in the absence of Hh ligand, where binding of Hh to Ptc allows activation of gene expression through the downstream transcription factor Cubitus interruptus (Ci). Ptc was inhibited within the dorsal compartment of the wing disc via RNAi, leaving the ventral compartment as an internal control (Fig. 1A). This resulted in an overactivation of the Hh pathway within the dorsal anterior region, which had a significantly slower decline of ATP levels after OxPhos inhibition (Fig. 1C). To further assess whether the Hh pathway is required for glycolysis in the wing disc, a dominant negative form of Ci was overexpressed within the dorsal compartment. Upon loss of Hh signaling in this region, ATP levels declined faster than within the ventral compartment after treatment with antimycin A. Altogether, the results presented here and in their previous work (4), reveal a positive feedback loop between Hh and glycolysis that is needed to couple energy production and patterning during development.

 

Figure 1. A) Ptc is inhibited only in the dorsal compartment of the wing disc, resulting in an overactivation of the Hh pathway in the dorsal anterior (DA) region (B). C) Increased Hh signaling leads to slower ATP depletion in the DA region of the wing disc after antimycin A treatment. (Adapted from Figure 3, Nellas et al. 2021)

 

Why I chose this preprint

This preprint utilizes a simple but powerful system, the Drosophila wing disc, to further study the interplay between metabolism and morphogen signaling, a topic where little is known. The results presented here add important and convincing evidence that developmental patterning events rely on metabolism, and vice versa. In addition, utilization of the FRET-based ATP sensor offers spatial information that is crucial when studying patterning events. Studies like this are helping to lay the foundation for other researchers exploring metabolic regulation during development.

Questions for the authors

  1. When assessing the kinetics of ATP depletion, in some instances the Half-life would show a significant difference between regions, whereas the Hill coefficient was not significantly different (e.g., Fig 3). Could the authors comment on whether there is a difference in reliability of readouts of kinetics?
  2. It looks like the dorsal portion of the wing disc that is expressing the CiDN (and therefore has reduced Hh signaling/glycolysis) is visibly smaller than the ventral WT portion of the wing disc. Did you see any abnormal outgrowth/increased growth within the dorsal anterior region of the PtcRNAi wing disc (which has increased Hh signaling/glycolysis)?
  3. A limited number of studies have made a connection between NAD+ metabolism and morphogen signaling (5,6). Considering the importance of NAD+ in glycolysis and OxPhos, do you think NAD+ levels could have an influence on Hh signaling in your system?

References:

  1. Oginuma, M., Moncuquet, P., Xiong, F., Karoly, E., Chal, J., Guevorkian, K., & Pourquié, O. (2017). A Gradient of Glycolytic Activity Coordinates FGF and Wnt Signaling during Elongation of the Body Axis in Amniote Embryos. Developmental Cell, 40(4), 342–353.e10.
  2. Bulusu, V., Prior, N., Snaebjornsson, M. T., Kuehne, A., Sonnen, K. F., Kress, J., Stein, F., Schultz, C., Sauer, U., & Aulehla, A. (2017). Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development. Developmental Cell, 40(4), 331–341.e4.
  3. Bhattacharya, D., Azambuja, A. P., & Simoes-Costa, M. (2020). Metabolic Reprogramming Promotes Neural Crest Migration via Yap/Tead Signaling. Developmental Cell, 53(2), 199–211.e6.
  4. Spannl, S. et al. (2020). Glycolysis regulates Hedgehog signaling via the plasma membrane potential. The EMBO Journal, 39(21), p. e101767. doi: 10.15252/embj.2019101767.
  5. Piao, J., Tsuji, K., Ochi, H. et al. (2013). Sirt6 regulates postnatal growth plate differentiation and proliferation via Ihh signaling. Sci Rep, 3, 3022. https://doi.org/10.1038/srep03022.
  6. Lee, J., Kee, H. J., Min, S. et al. (2016). Integrated omics-analysis reveals Wnt-mediated NAD+ metabolic reprogramming in cancer stem-like cells. Oncotarget, 7(30), 48562–48576. https://doi.org/10.18632/oncotarget.10432

Tags: energy, fly wing, patterning

Posted on: 8th October 2021

doi: https://doi.org/10.1242/prelights.30782

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Author's response

Natalie Dye shared

  1. When assessing the kinetics of ATP depletion, in some instances the Half-life would show a significant difference between regions, whereas the Hill coefficient was not significantly different (e.g., Fig 3). Could the authors comment on whether there is a difference in reliability of readouts of kinetics?

Both values are needed to fully describe the kinetic profile and thus should be considered together. Neither is more reliable – rather, each provides different kind of information. The half-life describes the time needed to achieve the halfway point between max and base (A), whereas the Hill coefficient describes the slope of the fitted curve at the half-value (B). For example, in the case of the PtcRNAi discs treated with Antimycin, only the half-life changes significantly but not the Hill coefficient. Thus, the affected tissue is able to maintain steady state levels of ATP for longer than in the internal control, but when the levels drop, they do so with a similar rate. In contrast, in the CiDN case, ATP levels begin to decline sooner, and they drop even faster than in the control tissue. It will be interesting in the future to further investigate the underlying mechanisms, using both experiments and mathematical modeling.

  1. It looks like the dorsal portion of the wing disc that is expressing the CiDN (and therefore has reduced Hh signaling/glycolysis) is visibly smaller than the ventral WT portion of the wing disc. Did you see any abnormal outgrowth/increased growth within the dorsal anterior region of the PtcRNAi wing disc (which has increased Hh signaling/glycolysis)?

Indeed, we do see that the dorsal compartment that is overexpressing CiDN is smaller than the control (ventral) compartment, although we do not report a quantification of this result. When PtcRNAi is driven in the dorsal compartment, we see overgrowth and a higher mitotic density (shown in Fig S5). Interestingly, however, we do not observe a significant difference in mitotic density between the anterior and posterior regions of the dorsal compartment, suggesting that the effects of PtcRNAi on metabolism can be uncoupled from its effects on proliferation.

 

  1. A limited number of studies have made a connection between NAD+ metabolism and morphogen signaling (5,6). Considering the importance of NAD+ in glycolysis and OxPhos, do you think NAD+ levels could have an influence on Hh signaling in your system?

We don’t know, as we have not explored the NAD+/NADH ratio or the overall redox state in the wing disc, so we can only speculate. There are some very interesting older reports in the literature of spatial patterns of NADPH producing enzymes in the wing. Colorimetric enzyme activity assays performed on wing disc explants indicate that G6PD is active in the DV but not the AP boundary, whereas IDH is active everywhere in the wing disc except for the AP boundary (Cunningham et al. 1983, Kuhn and Cunningham 1986). It would be interesting to look at the spatial expression and/or activity of NAD kinases and NADP phosphatases that interconvert these redox cofactors, as well as to look for any functional interaction with the Hh pathway.

Refs:

Cunningham GN, Smith NM, Makowski MK, Kuhn DT. Enzyme patterns in D. melanogaster imaginal discs: distribution of glucose-6-phosphate and 6-phosphogluconate dehydrogenase. Mol Gen Genet. 1983;191(2):238-43. doi: 10.1007/BF00334820. PMID: 6413822.

Kuhn DT, Cunningham GN. Isocitrate dehydrogenase in D. melanogaster imaginal discs: pattern development and alteration by homoeotic mutant genes. Dev Genet. 1986;7(1):21-34. doi: 10.1002/dvg.1020070103. PMID: 2899465.

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