Gcn5 – mTORC1 – TFEB signalling axis mediated control of autophagy regulates Drosophila blood cell homeostasis

  1. AR Arjun
  2. Suraj Math
  3. Laxmi Kata
  4. Rohan Jayant Khadilkar

  • Curated by eLife

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    This manuscript shows that manipulating the expression of the histone acetyltransferase Gcn5 affects blood cell homeostasis in the Drosophila larval hematopoietic organ. The data suggest a link between autophagy and the mTOR pathway, as could be expected from the literature. The authors use several genetic manipulations as well as some chemical modulators to generate solid evidence supporting most of their conclusions, but some of the analyses are inadequate and would benefit from improvement.

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Abstract

Blood progenitors are regulated by a variety of signals from their environment. In the Drosophila lymph gland (LG), the Posterior Signalling Center (PSC) acts as a stem cell niche striking a balance between progenitors and differentiated blood cells. While the response of blood progenitors to extrinsic signals is well characterized, their ability to respond to cell intrinsic cues is unexplored. Autophagy is one such intrinsic cellular process that maintains cellular homeostasis by removing unnecessary or dysfunctional cell components through autophagic degradation and recycling. Here, we show that autophagy plays a critical role in regulating blood cell homeostasis in the lymph gland. General control non-derepressible 5 (Gcn5), a histone acetyltransferase is expressed in all the cellular subsets of the LG and modulation of Gcn5 levels in various cellular subsets of the LG perturbs LG homeostasis. Gcn5 through its known non-histone acetylation target, TFEB controls autophagic flux thereby regulating hematopoiesis. Additionally, we demonstrate that modulation of mTORC1 activity can perturb hematopoiesis. We show that Gcn5 acts as a nutrient sensor and mTORC1 activity regulates Gcn5. mTORC1 over-rides the effect exerted by Gcn5 in regulating LG hematopoiesis. Together, our findings indicate that Gcn5 – mTORC1 – TFEB signaling axis mediated control of autophagy is required for maintaining blood cell homeostasis in Drosophila .

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  1. Version published to 10.7554/elife.91615.1 on eLife
  2. Version published to 10.7554/elife.91615 on eLife
  3. eLife

    eLife assessment

    This manuscript shows that manipulating the expression of the histone acetyltransferase Gcn5 affects blood cell homeostasis in the Drosophila larval hematopoietic organ. The data suggest a link between autophagy and the mTOR pathway, as could be expected from the literature. The authors use several genetic manipulations as well as some chemical modulators to generate solid evidence supporting most of their conclusions, but some of the analyses are inadequate and would benefit from improvement.

  4. eLife

    Reviewer #1 (Public Review):

    In their manuscript, Arjun et al. investigate the role of the histone acetyltransferase Gcn5 in the control of drosophila blood cell homeostasis in the larval lymph gland. They use gcn5 zygotic mutants as well as targeted knock-down and over-expression of Gcn5 in various lymph gland populations to show that these modulations impact (in a rather haphazard manner) niche cell number, blood cell progenitor maintenance, plasmatocyte differentiation, crystal cell differentiation or DNA damage accumulation. Their results suggest that Gcn5 controls autophagy and they show that decreasing the expression of the autophagy machinery increases blood cell differentiation. Using drugs to modulate the mTOR pathway, they conclude that Gcn5 levels are regulated by mTOR but that the impact of this pathway on blood cell homeostasis can override Gcn5 function.

    While the authors did a lot of experiments and good quantifications of the blood cell phenotypes, many results do not make much sense or do not bring valuable information about Gcn5 mode of action. Several conclusions of the manuscripts are not backed by solid data (e.g. that Gcn5 action is mediated by TFEB and the autophagy machinery) and different aspects of the literature are not well taken into consideration. Some results (such as the validation of the knockdown and overexpression of Gcn5) seem flawed. There are some concerns about the results obtained with gcn5 zygotic mutants and an interpretation of the phenotypes observed upon manipulation of Gcn5 expression in different cell types is missing.

    Important revisions are needed to improve the quality of the manuscript and confirm the authors' findings.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:
    Drosophila hematopoiesis has been shown to be governed by a number of signaling pathways such as JAK/STAT and Dpp. This important study shows the role of nutrient sensing and autophagy in determining blood cell differentiation. The authors show that General control non-derepressible 5 (Gcn5), a histone acetyltransferase affects blood cell differentiation. Gcn5 also negatively regulates autophagy through its effector TFEB which directly regulates autophagy genes. The authors also show that mTORC1 modulates Gcn5 levels and through it, TFEB activity thus acting as a fine-tuning mechanism that maintains optimal levels of autophagy.

    Strengths:
    The main strength of the work lies in the interesting finding that cellular metabolic processes such as autophagy have a direct role in blood cell differentiation and has the potential to be of interest to those working on vertebrate haematopoiesis as well. The report has generated intriguing data, using promoters specific for sub-sections of the lymph gland, that different cellular subsets of the lymph gland contribute differently towards haematopoiesis, but this is not followed up in detail and the final conclusions are derived from a combination of whole lymph gland perturbations as well as those from specific promoters.

    Weaknesses:
    1. Gc5 seems to be expressed throughout the lymph gland but modulating it in the subsections does not have the same result. It is very striking that the knockdown of Gcn5 in the prohemocyte population does not have an effect on differentiation whereas overexpression does. The modulations of Gcn5 in PSC also have variable effects across hemocyte subpopulations which is not explored in the manuscript. Interestingly, also the domain deletion constructs show a differential effect on blood cell differentiation when altered solely in the prohemocytes which is not explained. While Gcn5 can be seen in all sections of the lymph gland in the first figure, under the HHLT-Gal4 and Hml-Gal4, Gcn5 looks cytoplasmic and almost completely excluded from the nucleus strikingly unlike Gcn5 expression under the Collier-Gal4 and Dome-Gal4. The rest of the experiments in the manuscript are done with multiple promoters, with autophagy flux measured by modulating Gcn5 with a pan hemocyte promoter, but the mTORC1-Gcn5 axis is explored using chemical modulators which affect the whole of the lymph gland (Fig7) or using two pro-hemocyte promoters (Fig8).

    2. The knockdown of Gcn5 seems to affect the gland size (A compared to B and C). Since mTORC1 is a central regulator of cell size, it is possible that some of the effects seen in these knockdowns are potentially through mTORC1 affecting size suggesting that the signalling axis between mTORC1 and Gcn5 might not be a one-way axis as suggested in Figure 9. Also, this would mean that in experiments where absolute cell counts of crystal cells or niche cells are used to assess blood cell differentiation, further analysis to consider total cell numbers in the lymph gland would strengthen the manuscript.

    3. A genetic manipulation of mTORC1 specifically in the pro hemocytes would strengthen the role of mTORC1 in the pathway rather than the chemical modulation which affects the whole of the lymph gland.

  6. Version published to 10.1101/2023.07.04.547637v2 on bioRxiv
  7. Version published to 10.1101/2023.07.04.547637v1 on bioRxiv