Fat body-derived cytokine Upd2 controls disciplined migration of tracheal stem cells in Drosophila
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eLife assessment
This valuable study investigates how inter-organ communication between the tracheal stem cells and the fat body plays a key role in the directed migration of tracheal stem cells in Drosophila pupae. While the experimental data are extensive and complementary, the evidence presented to substantiate some of the conclusions appears incomplete and requires further clarification and additional experiments. The work would be of interest to researchers in the fields of developmental biology and cancer biology.
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Abstract
Coordinated activation and directional migration of adult stem cells are essential for maintaining tissue homeostasis. Drosophila tracheal progenitors are adult stem cells that migrate posteriorly along the dorsal trunk to replenish degenerating branches that disperse the FGF mitogen. However, it is currently unknown how the overall anterior-to-posterior directionality of such migration is controlled. Here we show that individual progenitor cells migrate together in a concerted, disciplined manner, a behavior that is dependent on the neighboring fat body. We identify the fat body-derived cytokine, Upd2, in targeting and inducing JAK/STAT signaling in tracheal progenitors to maintain their directional migration. Perturbation of either Upd2 production in fat body or JAK/STAT signaling in trachea causes aberrant bidirectional migration of tracheal progenitors. We show that JAK/STAT signaling promotes the expression of genes involved in planar cell polarity leading to asymmetric localization of Fat in progenitor cells. We provide evidence that Upd2 transport requires Rab5- and Rab7-mediated endocytic sorting and Lbm-dependent vesicle trafficking. Our study thus uncovers an inter-organ communication in the control of disciplined migration of tracheal progenitor cells, a process that requires vesicular trafficking of fat body-derived cytokine Upd2 and JAK/STAT signaling-mediated activation of PCP genes.
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eLife assessment
This valuable study investigates how inter-organ communication between the tracheal stem cells and the fat body plays a key role in the directed migration of tracheal stem cells in Drosophila pupae. While the experimental data are extensive and complementary, the evidence presented to substantiate some of the conclusions appears incomplete and requires further clarification and additional experiments. The work would be of interest to researchers in the fields of developmental biology and cancer biology.
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Reviewer #1 (Public review):
Summary:
In this manuscript, Dong et al. study the directed cell migration of tracheal stem cells in Drosophila pupae. The migration of these cells which are found in two nearby groups of cells normally happens unidirectionally along the dorsal trunk towards the posterior. Here, the authors study how this directionality is regulated. They show that inter-organ communication between the tracheal stem cells and the nearby fat body plays a role. They provide compelling evidence that Upd2 production in the fat body and JAK/STAT activation in the tracheal stem cells play a role. Moreover, they show that JAK/STAT signalling might induce the expression of apicobasal and planar cell polarity genes in the tracheal stem cells which appear to be needed to ensure unidirectional migration. Finally, the authors suggest …
Reviewer #1 (Public review):
Summary:
In this manuscript, Dong et al. study the directed cell migration of tracheal stem cells in Drosophila pupae. The migration of these cells which are found in two nearby groups of cells normally happens unidirectionally along the dorsal trunk towards the posterior. Here, the authors study how this directionality is regulated. They show that inter-organ communication between the tracheal stem cells and the nearby fat body plays a role. They provide compelling evidence that Upd2 production in the fat body and JAK/STAT activation in the tracheal stem cells play a role. Moreover, they show that JAK/STAT signalling might induce the expression of apicobasal and planar cell polarity genes in the tracheal stem cells which appear to be needed to ensure unidirectional migration. Finally, the authors suggest that trafficking and vesicular transport of Upd2 from the fat body towards the tracheal cells might be important.
Strengths:
The manuscript is well written. This novel work demonstrates a likely link between Upd2-JAK/STAT signalling in the fat body and tracheal stem cells and the control of unidirectional cell migration of tracheal stem cells. The authors show that hid+rpr or Upd2RNAi expression in a fat body or Dome RNAi, Hop RNAi, or STAT92E RNAi expression in tracheal stem cells results in aberrant migration of some of the tracheal stem cells towards the anterior. Using ChIP-seq as well as analysis of GFP-protein trap lines of planar cell polarity genes in combination with RNAi experiments, the authors show that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells which appear to be needed for unidirectional migration. Moreover, the authors hypothesise that extracellular vesicle transport of Upd2 might be involved in this Upd2-JAK/STAT signalling in the fat body and tracheal stem cells, which, if true, would be quite interesting and novel.
Overall, the work presented here provides some novel insights into the mechanism that ensures unidirectional migration of tracheal stem cells that prevents bidirectional migration. This might have important implications for other types of directed cell migration in invertebrates or vertebrates including cancer cell migration.
Weaknesses:
It remains unclear to what extent Upd2-JAK/STAT signalling regulates unidirectional migration. While there seems to be a consistent phenotype upon genetic manipulation of Upd2-JAK/STAT signalling and planar cell polarity genes, as in the aberrant anterior migration of a fraction of the cells, the phenotype seems to be rather mild, with the majority of cells migrating towards the posterior.
While I am not an expert on extracellular vesicle transport, the data presented here regarding Upd2 being transported in extracellular vesicles do not appear to be very convincing.
Major comments:
(1) The graphs showing the quantification of anterior (and in some cases also posterior migration) are quite confusing. E.g. Figure 1F (and 5E and all others): These graphs are difficult to read because the quantification for the different conditions is not shown separately. E.g. what is the migration distance for Fj RNAi anterior at 3h in Fig5E? Around -205micron (green plus all the other colors) or around -70micron (just green, even though the green bar goes to -205micron). If it's -205micron, then the images in C' or D' do not seem to show this strong phenotype. If it's around -70, then the way the graph shows it is misleading, because some readers will interpret the result as -205.
Moreover, it's also not clear what exactly was quantified and how it was quantified. The details are also not described in the methods. It would be useful, to mark with two arrowheads in the image (e.g. 5 A' -D') where the migration distance is measured (anterior margin and point zero).
Overall, it would be better, if the graph showed the different conditions separately. Also, n numbers should be shown in the figure legend for all graphs.
(2) Figure 2-figure supplement 1: C-L and M: From these images and graph it appears that Upd2 RNAi results in no aberrant anterior migration. Why is this result different from Figures 2D-F where it does?
(3) Figure 5F: The data on the localisation of planar cell polarity proteins in the tracheal stem cell group is rather weak. Figure 5G and J should at least be quantified for several animals of the same age for each genotype. Is there overall more Ft-GFP in the cells on the posterior end of the cell group than on the opposite side? Or is there a more classic planar cell polarity in each cell with Ft-GFP facing to the posterior side of the cell in each cell? Maybe it would be more convincing if the authors assessed what the subcellular localisation of Ft is through the expression of Ft-GFP in clones to figure out whether it localises posteriorly or anteriorly in individual cells.
(4) Regarding the trafficking of Upd2 in the fat body, is it known, whether Grasp65, Lbm, Rab5, and 7 are specifically needed for extracellular vesicle trafficking rather than general intracellular trafficking? What is the evidence for this?
(5) Figure 8A-B: The data on the proximity of Rab5 and 7 to the Upd2 blobs are not very convincing.
(6) The authors should clarify whether or not their work has shown that "vesicle-mediated transport of ligands is essential for JAK/STAT signaling". In its current form, this manuscript does not appear to provide enough evidence for extracellular vesicle transport of Upd2.
(7) What is the long-term effect of the various genetic manipulations on migration? The authors don't show what the phenotype at later time points would be, regarding the longer-term migration behaviour (e.g. at 10h APF when the cells should normally reach the posterior end of the pupa). And what is the overall effect of the aberrant bidirectional migration phenotype on tracheal remodelling?
(8) The RNAi experiments in this manuscript are generally done using a single RNAi line. To rule out off-target effects, it would be important to use two non-overlapping RNAi lines for each gene.
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Reviewer #2 (Public review):
Summary:
This work by Dong and colleagues investigates the directed migration of tracheal stem cells in Drosophila pupae, essential for tissue homeostasis. These cells, found in two nearby groups, migrate unidirectionally along the dorsal trunk towards the posterior to replenish degenerating branches that disperse the FGF mitogen. The authors show that inter-organ communication between tracheal stem cells and the neighboring fat body controls this directionality. They propose that the fat body-derived cytokine Upd2 induces JAK/STAT signaling in tracheal progenitors, maintaining their directional migration. Disruption of Upd2 production or JAK/STAT signaling results in erratic, bidirectional migration. Additionally, JAK/STAT signaling promotes the expression of planar cell polarity genes, leading to …
Reviewer #2 (Public review):
Summary:
This work by Dong and colleagues investigates the directed migration of tracheal stem cells in Drosophila pupae, essential for tissue homeostasis. These cells, found in two nearby groups, migrate unidirectionally along the dorsal trunk towards the posterior to replenish degenerating branches that disperse the FGF mitogen. The authors show that inter-organ communication between tracheal stem cells and the neighboring fat body controls this directionality. They propose that the fat body-derived cytokine Upd2 induces JAK/STAT signaling in tracheal progenitors, maintaining their directional migration. Disruption of Upd2 production or JAK/STAT signaling results in erratic, bidirectional migration. Additionally, JAK/STAT signaling promotes the expression of planar cell polarity genes, leading to asymmetric localization of Fat in progenitor cells. The study also indicates that Upd2 transport depends on Rab5- and Rab7-mediated endocytic sorting and Lbm-dependent vesicle trafficking. This research addresses inter-organ communication and vesicular transport in the disciplined migration of tracheal progenitors.
Strengths:
This manuscript presents extensive and varied experimental data to show a link between Upd2-JAK/STAT signaling and tracheal progenitor cell migration. The authors provide convincing evidence that the fat body, located near the trachea, secretes vesicles containing the Upd2 cytokine. These vesicles reach tracheal progenitors and activate the JAK-STAT pathway, which is necessary for their polarized migration. Using ChIP-seq, GFP-protein trap lines of planar cell polarity genes, and RNAi experiments, the authors demonstrate that STAT92E likely regulates the transcription of planar cell polarity genes and some apicobasal cell polarity genes in tracheal stem cells, which seem to be necessary for unidirectional migration.
Weaknesses:
Directional migration of tracheal progenitors is only partially compromised, with some cells migrating anteriorly and others maintaining their posterior migration.
Additionally, the authors do not examine the potential phenotypic consequences of this defective migration.It is not clear whether the number of tracheal progenitors remains unchanged in the different genetic conditions. If there are more cells, this could affect their localization rather than migration and may change the proposed interpretation of the data.
Upd2 transport by vesicles is not convincingly shown.
Data presentation is confusing and incomplete.
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Reviewer #3 (Public review):
Summary:
Dong et al tackle the mechanism leading to polarized migration of tracheal progenitors during Drosophila metamorphosis. This work fits in the stem cell research field and its crucial role in growth and regeneration. While it has been previously reported by others that tracheal progenitors migrate in response to FGF and Insulin signals emanating from the fat body in order to regenerate tracheal branches, the authors identified an additional mechanism involved in the communication of the fat body and tracheal progenitors.
Strengths:
The data presented were obtained using a wide range of complementary techniques combining genetics, molecular biology, quantitative, and live imaging techniques. The authors provide convincing evidence that the fat body, found in close proximity to the trachea, secrete …
Reviewer #3 (Public review):
Summary:
Dong et al tackle the mechanism leading to polarized migration of tracheal progenitors during Drosophila metamorphosis. This work fits in the stem cell research field and its crucial role in growth and regeneration. While it has been previously reported by others that tracheal progenitors migrate in response to FGF and Insulin signals emanating from the fat body in order to regenerate tracheal branches, the authors identified an additional mechanism involved in the communication of the fat body and tracheal progenitors.
Strengths:
The data presented were obtained using a wide range of complementary techniques combining genetics, molecular biology, quantitative, and live imaging techniques. The authors provide convincing evidence that the fat body, found in close proximity to the trachea, secrete vesicles containing the Upd2 cytokine that reach tracheal progenitors leading to JAK-STAT pathway activation, which is required for their polarized migration. In addition, the authors show that genes regulating planar cell polarity are also involved in this inter-organ communication.
Weaknesses:
(1) Affecting this inter-organ communication leads to a quite discrete phenotype where polarized migration of tracheal progenitors is partially compromised. The study lacks data showing the consequences of this phenotype on the final trachea morphology, function, and/or regeneration capacities at later pupal and adult stages. This could potentially increase the significance of the findings.
(2) The conclusions of this paper are mostly well supported by data, but some aspects of data acquisition and analysis need to be clarified and corrected, such as recurrent errors in plotting of tracheal progenitor migration distance that mislead the reader regarding the severity of the phenotype.
(3) The number of tracheal progenitors should be assessed since they seem to be found in excess in some genetic conditions that affect their behavior. A change in progenitor number could lead to crowding, thus affecting their localization rather than migration capacities, thereby changing the proposed interpretation. In addition, the authors show data suggesting a reduced progenitor migration speed when the fat body is affected, which would also be consistent with a crowding of progenitors.
(4) The authors claim that tracheal progenitors display a polarized distribution of PCP proteins that is controlled by JAK-STAT signaling. However, this conclusion is made from a single experiment that is not quantified and for which there is no explanation of how the plot profile measurements were performed. It also seems that this experiment was done only once. Altogether, this is insufficient to support the claim. Finally, a quantification of the number of posterior edges presenting filopodia rather than the number of filopodia at the anterior and posterior leading edges would be more appropriate.
(5) The authors demonstrate that Upd2 is transported through vesicles from the fat body to the tracheal progenitors where they propose they are internalized. Since the Upd2 receptor Dome ligand binding sites are exposed to the extracellular environment, it is difficult to envision in the proposed model how Upd2 would be released from vesicles to bind Dome extracellularly and activate the JAK-STAT pathway. Moreover, data regarding the mechanism of the vesicular transport of Upd2 are not fully convincing since the PLA experiments between Upd2 and Rab5, Rab7, and Lbm are not supported by proper positive and negative controls and co-immunoprecipitation data in the main figure do not always correlate to the raw data.
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