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mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis

Marie Clémot, Cecilia D’Alterio, Alexa Kwang, D. Leanne Jones

Preprint posted on 25 July 2022 https://www.biorxiv.org/content/10.1101/2022.07.25.501357v1.full

One signal to differentiate them both and in timed development bind them: Tor is required for germ cell differentiation and its coordination with supporting somatic cells in the Drosophila testis

Selected by Diego Sainz de la Maza

Background

Adult stem cells maintain tissue homeostasis by balancing self-renewal and differentiation. They reside in specialized microenvironments termed niches, where they receive supporting signaling molecules. Metabolism has been increasingly linked to cell fate decisions, but how metabolic cues are integrated with the signaling occurring in the stem cell niche is still unclear.

The Drosophila testis has a stem cell niche located at the apical tip of the tissue, which suports two different stem cell populations. Germline stem cells (GSCs) self-renew and give rise to gonialblasts, which undergo four uncomplete divisions and produce germ cysts consisting of 2, 4, 8 or 16 spermatogonia. Somatic stem cells are termed cyst stem cells, and give rise to cyst cells, which ensheath the germline and support it throughout its development.

Previous findings suggest that the testis is highly sensitive to metabolic perturbations. Firstly, citrate from the gut reaches the testis and is required for sperm maturation. Secondly, protein starvation causes the death of transit-amplifying germ cells. In addition, insulin signaling is required by supporting somatic cells to differentiate. And finally, previous work from the Jones Lab showed that depletion of the regulator of mitochondrial fusion Mitofusin induced loss of GSCs in a TOR-dependent manner.

The preprint I have chosen to highlight explores the effects of TOR signaling on the germline and the entire tissue in great detail.

Why I chose this study

Metabolism has often been regarded as a mere succession of chemical reactions aimed to either obtain energy or synthesize macromolecules. From that point of view, metabolism would be a downstream effector of cellular choices already taken at the transcriptional level.

However, it has been reported that increasing activity of specific metabolic pathways can affect cell proliferative capacity or influence cell fate choices. These observations raise the possibility that metabolism can also influence cellular decision making. Considering that cells reside in tissues and are continuously sensing their surrounding environment, it seems reasonable that nutrient availability and the subsequent activity of metabolic pathways has certain degree of crosstalk with signaling pathways and ultimately with cell behaviour.

As far as stem cells are concerned, they are involved in tremendously complex processes such as development or tissue homeostasis. One could predict that stem cells need to coordinate the production of new cells to a favourable nutritional or metabolic status, a very exciting field that has not been extensively explored.

This highlighted preprint explores the role of TOR in germline differentiation. I chose this preprint because previous work had already linked TOR activity as a requirement for differentiation of somatic cells. Do both the soma and the germline integrate metabolic cues through TOR signaling? Is TOR responsible for differentiation in both cases? In addition to the effects that TOR could have on both lineages autonomously, this preprint raises another important question, which is how the cell identity of a cell affects other surrounding cell types when they must differentiate in a coordinated manner, as can be observed in the testis.

Main findings

TOR is present and active in early germ cells

The authors studied the presence and activity of TOR across the germline. To study its location, they used a TOR::GFP fusion protein. They reported that GSCs and early germ cells had higher levels of TOR than differentiating spermatogonia, which had lower levels and were restricted to the nucleus. In order to check where TOR is active, they used an antibody against the phosphorylated form of a TOR target and detected its signal in a subset of GSCs and early germ cells. Thus, TOR is present mostly in early germ cells and is active in a subset of them. When germ cells differentiate, TOR levels decrease.

TOR activity is required in early germ cells for differentiation

The authors addressed the impact of TOR on germ cell identity by performing loss of function experiments. They knocked down TOR in the germline and observed an accumulation of early germ cells in the tissue. No phenotype was observed when TOR was knocked down in germ cells that had started their differentiation program. This suggests that TOR activity is required in early germ cells for their differentiation and once its differentiation program has started TOR is downregulated and no longer required.

TOR knockdown in early germ cells triggers differentiation of somatic cells

Finally, the authors asked if disruption of germ cell identity had a non-autonomous effect on the neighbouring somatic cells. They knocked down TOR in the germline and studied somatic cell identity. They observed that TOR knockdown in the germline significantly decreased the number of somatic cells expressing a marker for early somatic cells. They checked if somatic cells were lost to differentiation in this context and found that knockdown of TOR in the germline upregulated TOR activity in the soma and caused the premature appearance of differentiation markers in the somatic cells. These data suggests that TOR downregulation couples germ cell and soma differentiation.

In conclusion, these findings support a model where active TOR in the germline is required to start its differentiation program and once this is triggered TOR signaling is no longer required. This downregulation of TOR in the germline is sensed by the soma, which starts its differentiation program to support the germline.

Tags: differentiation, germ cells, stem cells, tor signalling

Posted on: 28 November 2022

doi: https://doi.org/10.1242/prelights.33199

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Discussion with the authors

Marie Clémot shared

Question 1. The authors find an interesting heterogeneous pattern of TOR activity across the germline. Can the authors speculate what could be the mechanism behind it? Could it be differential levels of signals arriving from the niche? Could this be related to the reported production of inhibitors of insulin signaling by the surrounding somatic stem cells?

Answer. That is an interesting thought. I think that we favour a different hypothesis. A similar patchy pattern of Tor activity was previously reported in developing wing discs. We think that pS6, the TOR target that we used as a readout for TOR activity, is cell cycle dependent and the heterogenous positivity reflects the proliferation status. We cannot rule out the effect of a signal from the niche sensed differently by the germ cells, but it is unlikely that this is due to the secretion of insulin inhibitors by somatic stem cells because that would only affect the germ cells adjacent to the niche and our pS6 staining labels several stages of the germ cell lineage, including GSCs and germ cells more distant from the hub.

Question 2. The loss of function experiments show that TOR downregulation disrupts differentiation of the germline and does not affect differentiation of the germ cells which have already started differentiating. This result suggests that active TOR is required at the onset of differentiation and then is progressively downregulated. It could be interesting to perform loss of function experiments that drive hyperactivation of the TOR pathway, such as knockdown of Tsc1, to better understand the requirement of TOR in the differentiation of the germline. In this context, is it possible that a block of differentiation would still take place since there would not be a downregulation in differentiating germ cells? Alternatively, could it also drive premature differentiation of the germline?

Answer. We think that hyperactivated TOR would cause premature differentiation of the germline. In a previous paper from the lab, we observed that increased TOR activity in early germ cells with perturbed mitochondrial function (knockdown of the mitochondrial fusion regulator mitofusin) was associated with a loss of GSCs, which could be rescued if TOR was inhibited. When we knockdown TSC1 or TSC2 in GSCs and early germ cells, we get the expected increase in active TOR, but the phenotype in the tissue is not striking. These data suggest that hyperactivated TOR would cause premature differentiation of the germline, but it could be difficult to see because TOR seems to be required on the onset of differentiation and then is no longer required. I tried to hyperactivate TOR in differentiating germ cells using a different driver but did not observe a phenotype, probably because this driver turns on too late, at a stage where the Tor kinase is no longer highly expressed.

Question 3. The authors show that TOR is involved in the proper differentiation timing of both the germline and the soma. Upon TOR downregulation in the germline, somatic cells express a differentiation marker prematurely. The authors speculate that TOR could be inhibiting the secretion of Spi from the germline to the soma, a reported differentiation signal for somatic cells. It could be relatively easy to test this hypothesis by using in situ hybridization in the tissue and either quantify or locate the expression of Spi both in control tissue and when TOR is downregulated in the germline. Are there any plans to do anything along these lines?

Answer. We focused on the possible impairment of Spi secretion at the protein level. In the preprint, we include data showing that knocking down TOR in the germline causes an accumulation of dysfunctional autolysosomes in early germ cells. We thought that there could be a defect in the germline to secret Spi and activate differentiation in somatic cells. Unfortunately, we could not find a good antibody or reporter. We didn’t study Spi at the transcriptional level, but it could be interesting to do so.

Question 4. Is there anything else that you would like to comment on?

Answer. I think that the preprint provides relevant data about the coordination of the differentiation of both soma and germline. I would like to follow up on that. But I was also very interested on the acumulation of autolysosomes when TOR is knocked down. They have a very big size, occupying part of the cytoplasm of the germ cells.

This is intriguing because when we knocked down genes involved in autophagy, we did not see a phenotype. Then, why would autophagy become dysfunctional when TOR is knocked down if does not seem to be required for normal differentiation?

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