The Drosophila hematopoietic niche assembles through collective cell migration controlled by neighbor tissues and Slit-Robo signaling

Reviewer #1 (Public Review):

Summary:

The study by Nelson et al. is focused on the formation of the Drosophila Posterior Signaling Center (PSC) which ultimately acts as a niche to support hematopoietic stem cells of the lymph gland (LG). Using a combination of genetics and live imaging, the authors show that PSC cells migrate as a tight collective and associate with multiple tissues during a trajectory that positions them at the posterior of the LG.
This is an important study that identifies Slit-Robo signaling as a regulator of PSC morphogenesis, and highlights the complex relationship of interacting cell types - PSC, visceral mesoderm (VM), and cardioblasts (CBs) - in the coordinated development of these three tissues during organ development. However, one point requiring clarification is the idea that PSC cells exhibit a collective cell migration; it is not clear that the cells are migrating rather than being pushed to a more dorsal position through dorsal closure and/or other similar large-scale embryo movement. This does not detract from the very interesting analysis of PSC morphogenesis as presented.

Strengths:

(1) Using the expression of Hid or Grim to ablate associated tissues, they find evidence that the VM and CB of the dorsal vessel affect PSC migration/morphology whereas the alary muscles do not. Slit is expressed by both VM and CBs, and therefore Slit-Robo signaling was investigated as PSCs express Robo.

(2) Using a combination of approaches, the authors convincingly demonstrate that Slit expression in the CBs and VM acts to support PSC positioning. A strength is the ability to knockdown slit levels in particular tissue types using the Gal4 system and RNAi.

(3) Although in the analysis of robo mutants, the PSC positioning phenotype is weaker in the individual mutants (robo1 and robo2) with only the double mutant (robo1,robo2) exhibiting a phenotype comparable to the slit RNAi. The authors make a reasonable argument that Slit-Robo signaling has an intrinsic effect, likely acting within PSCs because PSCs show a phenotype even when CBs do not (Figure 4G).

(4) New insight into dorsal vessel formation by VM is presented in Figure 4A, B, as loss of the VM can affect dorsal vessel morphogenesis. This result additionally points to the VM as important.

Weaknesses:

(1) The authors are cautioned to temper the result that Slit-Robo signaling is intrinsic to PSC since the loss of robo may affect other cell types (besides CBs and PSCs) to indirectly affect PSC migration/morphogenesis. In fact, in the robo2, robo1 mutant, the VM appears to be incorrectly positioned (Figure 4G).

(2) If possible, the authors should use RNAi to knockdown Robo1 and Robo2 levels specifically in the PSCs if a Gal4 is available; might Antp.Gal4 (Fig 1K) be useful? Even if knockdown is achieved in PSCs+CBs, this would be a better/complementary experiment to support the approach outlined in Figure 4D.

(3) Movies are hard to interpret, as it seems unclear that the PSCs actively migrate rather than being pushed/moved indirectly due to association with VM and CBs/dorsal vessel.

Reviewer #2 (Public Review):

The paper by Nelson KA, et al. explored the collective migration, coalescence, and positioning of the posterior signaling center (PSC) cells in Drosophila embryo. With live imaging, the authors observed the dynamic progress of PSC migration. Throughout this process, visceral mesoderm (VM), alary muscles (Ams), and cardioblasts (CBs) are in proximity to PSC. Genetic ablation of these tissues reveals the requirement for VM and CBs, but not AMs in this process. Genetic manipulations further demonstrated that Slit-Robo signaling was critical during PSC migration and positioning. While the genetic mechanisms of positioning the PSC were explored in much detail, including using live imaging, the functional consequence of mispositioning or (partial) absence of PSC cells has not been addressed, but would much increase the relevance of their findings. A few additional issues need to be addressed as well in this otherwise well-done study.

Major points:

(1) The only readout in their experiments is the relative correctness of PSC positioning. Importantly, what is the functional consequence if PSC is not properly positioned? This would be particularly important with robo-sli manipulations, where the PSC is present but some cells are misplaced. What is the consequence? Are the LGs affected, like the specification of their cell types, structure, and function? To address this for at least the robo-slit requirement in the PSC, it may be important to manipulate them directly in the PSC with a split Gal4 system, using Antp and Odd promoters.

(2) The densely, parallel aligned fibers in the part of Figure 1J seemed to be visceral mesoderm, but further up (dorsally) that may be epidermis. It is possible that the PSC migrate together with the epidermis? This should be addressed.

(3) Although the authors described the standards of assessing PSC positioning as "normal" or "abnormal", it is rather subtle at times and variable in the mutant or KD/OE examples. The criteria should be more clearly delineated and analyzed double-blind, also since this is the only readout. Further examples of abnormal positioning in supplementary figures would also help.

(4) The Discussion is very lengthy and should shortened.

Reviewer #3 (Public Review):

Summary:

This work is a detailed and thorough analysis of the morphogenesis of the posterior signaling center (PSC), a hematopoietic niche in the Drosophila larva. Live imaging is performed from the stage of PSC determination until the appearance of a compact lymph gland and PSC in the stage 16 embryo. This analysis is combined with genetic studies that clarify the involvement of adjacent tissue, including the visceral mesoderm, alary muscle, and cardioblasts/dorsal vessels. Lastly, the Slit/Robo signaling system is clearly implicated in the normal formation of the PSC.

Strengths:

The data are clearly presented, well documented, and fully support the conclusions drawn from the different experiments. The manuscript differs in character from the mainstay of "big data" papers (for example, no sets of single-cell RNAseq data of, for instance, PSC cells with more or less Slit input, are offered), but what it lacks in this regard, it makes up in carefully planned and executed visualizations and genetic manipulations.

Weaknesses:

A few suggestions concerning improvement of the way the story is told and contextualized.

(1) The minute cluster of PSC progenitors (5 or so cells per side) is embedded (as known before and shown nicely in this study) in other "migrating" cell pools, like the cardioblasts, pericardial cells, lymph gland progenitors, alary muscle progenitors. These all appear to move more or less synchronously. What should also be mentioned is another tissue, the dorsal epidermis, which also "moves" (better: stretches?) towards the dorsal midline during dorsal closure. Would it be reasonable to speculate (based on previously published data) that without the force of dorsal closure, operating in the epidermis, at least the lateral>medial component of the "migration" of the PSC (and neighboring tissues) would be missing? If dorsal closure is blocked, do essential components of PSC and lymph gland morphogenesis (except for the coming-together of the left and right halves) still occur? Are there any published data on this?

(2) Along similar lines: the process of PSC formation is characterized as "migration". To be fair: the authors bring up the possibility that some of the phenotypes they observe could be "passive"/secondary: "Thus, it became important to test whether all PSC phenotypes might be 'passive', explained by PSC attachment to a malforming dorsal vessel. Alternatively, the PSC defects could reflect a requirement for Robo activation directly in PSC cells." And the issue is resolved satisfactorily. But more generally, "cell migration" implies active displacement (by cytoskeletal forces) of cells relative to a substrate or to their neighbors (like for example migration of hemocytes). This to me doesn't seem really clearly to happen here for the dorsal mesodermal structures. Couldn't one rather characterize the assembly of PSC, lymph gland, pericardial cells, and dorsal vessel in terms of differential adhesion, on top of a more general adhesion of cells to each other and the epidermis, and then dorsal closure as a driving force for cell displacement? The authors should bring in the published literature to provide a background that does (or does not) justify the term "migration".

(3) That brings up the mechanistic centerpiece of this story, the Slit/Robo system. First: I suggest adding more detailed data from the study by Morin-Poulard et al 2016, in the Introduction, since these authors had already implicated Slit-Robo in PSC function and offered a concrete molecular mechanism: "vascular cells produce Slit that activates Robo receptors in the PSC. Robo activation controls proliferation and clustering of PSC cells by regulating Myc, and small GTPase and DE-cadherin activity, respectively". As stated in the Discussion: the mechanism of Slit/Robo action on the PSC in the embryo is likely different, since DE-cadherin is not expressed in the embryonic PSC; however, it maybe not be THAT different: it could also act on adhesion between PSC cells themselves and their neighbors. What are other adhesion proteins that appear in the late lateral mesodermal structures? Could DN-cadherin or Fasciclins be involved?

Author response:

eLife assessment

This study presents valuable findings on the role of a well-studied signal transduction pathway, the Slit/Robo system, in the context of the assembly of the hematopoietic niche in the Drosophila embryo. The evidence supporting the claims of the authors is solid. However, one aspect that needs attention is whether the cells are migrating and not being pushed to a more dorsal position through dorsal closure and/or other similar large-scale embryo movement. This does not detract from the very interesting analysis of PSC morphogenesis and will interest developmental biologists working on molecular mechanisms of tissue morphogenesis.

We appreciate the thoughtful and quite useful comments provided by each of the referees. Our responses are noted below each referee’s comment.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The study by Nelson et al. is focused on the formation of the Drosophila Posterior Signaling Center (PSC) which ultimately acts as a niche to support hematopoietic stem cells of the lymph gland (LG). Using a combination of genetics and live imaging, the authors show that PSC cells migrate as a tight collective and associate with multiple tissues during a trajectory that positions them at the posterior of the LG.

This is an important study that identifies Slit-Robo signaling as a regulator of PSC morphogenesis, and highlights the complex relationship of interacting cell types - PSC, visceral mesoderm (VM), and cardioblasts (CBs) - in the coordinated development of these three tissues during organ development. However, one point requiring clarification is the idea that PSC cells exhibit a collective cell migration; it is not clear that the cells are migrating rather than being pushed to a more dorsal position through dorsal closure and/or other similar large-scale embryo movement. This does not detract from the very interesting analysis of PSC morphogenesis as presented.

Since each referee asked for clarification concerning collective cell migration, we present a combined response further below, placed after the comments from Reviewer #3.

Strengths:

(1) Using the expression of Hid or Grim to ablate associated tissues, they find evidence that the VM and CB of the dorsal vessel affect PSC migration/morphology whereas the alary muscles do not. Slit is expressed by both VM and CBs, and therefore Slit-Robo signaling was investigated as PSCs express Robo.

(2) Using a combination of approaches, the authors convincingly demonstrate that Slit expression in the CBs and VM acts to support PSC positioning. A strength is the ability to knockdown slit levels in particular tissue types using the Gal4 system and RNAi.

(3) Although in the analysis of robo mutants, the PSC positioning phenotype is weaker in the individual mutants (robo1 and robo2) with only the double mutant (robo1,robo2) exhibiting a phenotype comparable to the slit RNAi. The authors make a reasonable argument that Slit-Robo signaling has an intrinsic effect, likely acting within PSCs because PSCs show a phenotype even when CBs do not (Figure 4G).

(4) New insight into dorsal vessel formation by VM is presented in Figure 4A, B, as loss of the VM can affect dorsal vessel morphogenesis. This result additionally points to the VM as important.

Weaknesses:

(1) The authors are cautioned to temper the result that Slit-Robo signaling is intrinsic to PSC since the loss of robo may affect other cell types (besides CBs and PSCs) to indirectly affect PSC migration/morphogenesis. In fact, in the robo2, robo1 mutant, the VM appears to be incorrectly positioned (Figure 4G).

We have reexamined our wording in the relevant Results section and, given that this referee agrees that we, “make a reasonable argument that Slit-Robo signaling has an intrinsic effect, likely acting within PSCs because PSCs show a phenotype even when CBs do not (Figure 4G)”, it was not clear how we might temper our conclusions more. Given that PSC cells express Robo1 and Robo2, and that the Vm does not contact the PSC, our ‘reasonable argument’ appears fair and parsimonious. Since we agree with the referee that a reader should be made as aware as possible of alternatives, we will add a comment to the Discussion, reminding the reader of the possibility of a secondary defect.

(2) If possible, the authors should use RNAi to knockdown Robo1 and Robo2 levels specifically in the PSCs if a Gal4 is available; might Antp.Gal4 (Fig 1K) be useful? Even if knockdown is achieved in PSCs+CBs, this would be a better/complementary experiment to support the approach outlined in Figure 4D.

While we agree that PSC-specific knockdown of Robo1 and Robo2 simultaneously would be ideal, this is not possible. First, the most-effective UAS-RNAi transgenes (that is, those in a Valium 20 backbone) are both integrated at the same chromosomal position; these cannot be simultaneously crossed with a GAL4 transgenic line to attempt double knock down. Additionally, as with all RNAi approaches that must rely on efficient knockdown over the rapid embryonic period, even having facile access to the above does not ensure the RNAi approach will cause as effective depletion as the genetic null condition that we use. Second, as the referee concedes, there is no embryonic PSC-specific GAL4. The proposed use of Antp-GAL4 would cause knockdown in many tissues (PSC, CB, Vm, epidermis and amnioserosa). This would lead to a reservation similar to that caused by our use of the straight genetic double mutant, as regards potential indirect requirement for Robo function.

(3) Movies are hard to interpret, as it seems unclear that the PSCs actively migrate rather than being pushed/moved indirectly due to association with VM and CBs/dorsal vessel.

First, the Vm does not directly contact the PSC, so it cannot be pushing the PSC dorsally. We will re-examine our text to be certain to make this clear. Second, in our analysis of bin mutants, which lack Vm, LGs and PSCs are able to reach the dorsal midline region in the absence of Vm. Finally, please see our response to Reviewer #3, point 2, for why we maintain that PSC cells are “migrating” even though some PSC cells are attached to CBs.

Reviewer #2 (Public Review):

The paper by Nelson KA, et al. explored the collective migration, coalescence, and positioning of the posterior signaling center (PSC) cells in Drosophila embryo. With live imaging, the authors observed the dynamic progress of PSC migration. Throughout this process, visceral mesoderm (VM), alary muscles (Ams), and cardioblasts (CBs) are in proximity to PSC. Genetic ablation of these tissues reveals the requirement for VM and CBs, but not AMs in this process. Genetic manipulations further demonstrated that Slit-Robo signaling was critical during PSC migration and positioning. While the genetic mechanisms of positioning the PSC were explored in much detail, including using live imaging, the functional consequence of mispositioning or (partial) absence of PSC cells has not been addressed, but would much increase the relevance of their findings. A few additional issues need to be addressed as well in this otherwise well-done study.

Major points:

(1) The only readout in their experiments is the relative correctness of PSC positioning. Importantly, what is the functional consequence if PSC is not properly positioned? This would be particularly important with robo-sli manipulations, where the PSC is present but some cells are misplaced. What is the consequence? Are the LGs affected, like the specification of their cell types, structure, and function? To address this for at least the robo-slit requirement in the PSC, it may be important to manipulate them directly in the PSC with a split Gal4 system, using Antp and Odd promoters.

We agree that the functional consequence of PSC mis-positioning is important and a relevant question to eventually address. However, virtually all markers and reagents used to assess the effect of the PSC on progenitor cells and their differentiated descendants are restricted to analyses carried out on the third larval instar - some three days after the experiments reported here. Most of the manipulated conditions in our work are no longer viable at this phase and, thus, addressing the functional consequences of a malformed PSC will require the field to develop new tools.

As we noted in the Introduction, the consistency with which the wildtype PSC forms as a coalesced collective at the posterior of the LG strongly suggests importance of its specific positioning and shape, as has now been found for other niches (citations in manuscript). Additionally, in the Discussion we mention the existence of a gap junction-dependent calcium signaling network in the PSC that is important for progenitor maintenance. Without continuity of this network amongst all PSC cells (under conditions of PSC mis-positioning), we strongly anticipate that the balance of progenitors to differentiated hemocytes will be mis-managed, either constitutively, and / or under immune challenge conditions.

Finally, to our knowledge, the tools do not exist to build a “split Gal4 system using Antp and Odd promoters”. The expression pattern observed using the genomic Antp-GAL4 line must be driven by endogenous enhancers–none of which have been defined by the field, and thus cannot be used in constructing second order drivers. Similarly, for odd skipped, in the embryo the extant Odd-GAL4 driver expresses only in the epidermis, with no expression in the embryonic LG. Thus, the cis regulatory element controlling Odd expression in the embryonic LG is unknown. In the future, the discovery of an embryonic PSC-specific driver will aid in addressing the specific functional consequences of PSC mis-positioning.

(2) The densely, parallel aligned fibers in the part of Figure 1J seemed to be visceral mesoderm, but further up (dorsally) that may be epidermis. It is possible that the PSC migrate together with the epidermis? This should be addressed.

See response to Reviewer #3.

(3) Although the authors described the standards of assessing PSC positioning as "normal" or "abnormal", it is rather subtle at times and variable in the mutant or KD/OE examples. The criteria should be more clearly delineated and analyzed double-blind, also since this is the only readout. Further examples of abnormal positioning in supplementary figures would also help.

We appreciate the Reviewer’s concern and acknowledge that the phenotypes we observed were indeed variable, and, at times subtle. As we show and discuss in the paper, our results revealed that the signaling requirements for proper PSC positioning are complex; this was favorably commented upon by Reviewer #1 (“...highlights the complex relationship of interacting cell types - PSC, visceral mesoderm (VM), and cardioblasts (CBs) - in the coordinated development of these three tissues during organ development.…”). We suspect the phenotypic variability is attributable to any number of biological differences such as heterogeneity of PSC cells and an accompanying difference in the timing of their competence to receive and respond to Slit-Robo signaling, the timing of release of Slit from CBs and Vm, number of cells in a given PSC, which PSC cells in the cluster respond to too little or too much signaling, and/or typical variability between organisms. Furthermore, PSC positioning analyses were conducted by two of the authors, who independently came to the same conclusions. For many of the manipulations double blinding was not possible since the genotype of the embryo was discernible due to the obvious phenotype of the manipulated tissue.

(4) The Discussion is very lengthy and should [be] shortened.

We will re-examine the prose and emphasize more conciseness, while maintaining clarity for the reader.

Reviewer #3 (Public Review):

Summary:

This work is a detailed and thorough analysis of the morphogenesis of the posterior signaling center (PSC), a hematopoietic niche in the Drosophila larva. Live imaging is performed from the stage of PSC determination until the appearance of a compact lymph gland and PSC in the stage 16 embryo. This analysis is combined with genetic studies that clarify the involvement of adjacent tissue, including the visceral mesoderm, alary muscle, and cardioblasts/dorsal vessels. Lastly, the Slit/Robo signaling system is clearly implicated in the normal formation of the PSC.

Strengths:

The data are clearly presented, well documented, and fully support the conclusions drawn from the different experiments. The manuscript differs in character from the mainstay of "big data" papers (for example, no sets of single-cell RNAseq data of, for instance, PSC cells with more or less Slit input, are offered), but what it lacks in this regard, it makes up in carefully planned and executed visualizations and genetic manipulations.

Weaknesses:

A few suggestions concerning improvement of the way the story is told and contextualized.

(1) The minute cluster of PSC progenitors (5 or so cells per side) is embedded (as known before and shown nicely in this study) in other "migrating" cell pools, like the cardioblasts, pericardial cells, lymph gland progenitors, alary muscle progenitors. These all appear to move more or less synchronously. What should also be mentioned is another tissue, the dorsal epidermis, which also "moves" (better: stretches?) towards the dorsal midline during dorsal closure. Would it be reasonable to speculate (based on previously published data) that without the force of dorsal closure, operating in the epidermis, at least the lateral>medial component of the "migration" of the PSC (and neighboring tissues) would be missing? If dorsal closure is blocked, do essential components of PSC and lymph gland morphogenesis (except for the coming-together of the left and right halves) still occur? Are there any published data on this?

Each of the Reviewers is interested in our response to this very relevant question, and, thus, we will address the issue en bloc here. First, we will add a Supplementary Figure showing that LG and CBs are still able to progress medially towards the dorsal midline when dorsal closure stalls. This rules out any major effect for the most prominent “large-scale embryo cell sheet movement” in positioning the PSC. Second, published work by Haack et. al. and Balaghi et. al. shows that CBs and leading edge epidermal cells are independently migratory, and we will add this context to the manuscript for the reader.

(2) Along similar lines: the process of PSC formation is characterized as "migration". To be fair: the authors bring up the possibility that some of the phenotypes they observe could be "passive"/secondary: "Thus, it became important to test whether all PSC phenotypes might be 'passive', explained by PSC attachment to a malforming dorsal vessel. Alternatively, the PSC defects could reflect a requirement for Robo activation directly in PSC cells." And the issue is resolved satisfactorily. But more generally, "cell migration" implies active displacement (by cytoskeletal forces) of cells relative to a substrate or to their neighbors (like for example migration of hemocytes). This to me doesn't seem really clearly to happen here for the dorsal mesodermal structures. Couldn't one rather characterize the assembly of PSC, lymph gland, pericardial cells, and dorsal vessel in terms of differential adhesion, on top of a more general adhesion of cells to each other and the epidermis, and then dorsal closure as a driving force for cell displacement? The authors should bring in the published literature to provide a background that does (or does not) justify the term "migration".

Before addressing this specifically, we remind readers of our response above that states the rationale ruling out large, embryo-scale movements, such as epidermal dorsal closure, in driving PSC positioning. So, how are PSC cells arriving at their reproducible position? This manuscript reports the first live-imaging of the PSC as it comes to be positioned in the embryo. We interpret these movies to suggest strongly that these cells are a ‘collective’ that migrates. Neither the data, nor we, are asserting that each PSC cell is ‘individually’ migrating to its final position. Rather, our data suggest that the PSC migrates as a collective. The most paradigmatic example of directed, collective cell migration, is of Drosophila ovarian border cells. That cell cluster is surrounded at all times by other cells (nurse cells, in that case), and for the collective to traverse through the tissue, the process requires constant remodeling of associations amongst the migrating cells in the collective (the border cells), as well as between cells in the collective and those outside of it (the nurse cells). In fact, the nurse cells are considered the substrate upon which border cells migrate. Note also that in collective border cell migration cells within the collective can switch neighbors, suggesting dynamic changes to cell associations and adhesions.

In our analysis, the PSC cells exhibit qualities reminiscent of the border cells, and thus we infer that the PSC constitutes a migratory cell collective. We also show in Figure 1H that PSC cells exhibit cellular extensions, and thus have a very active, intrinsic actin-based cytoskeleton. In fact, in Figure 1I, we point out that PSC cells shift position within the collective, which is not only a direct feature of migration, but also occurs within the border cell collective as that collective migrates. Additionally, the fact that the lateral-most PSC cells shift position in the collective while remaining a part of the collective–and they do this while executing net directional movement–makes a strong argument that the PSC is migratory, as no cell types other than PSCs are contacting the surfaces of those shifting PSC cells. Lastly, the Reviewer’s supposition that, rather than migration, dorsal mesoderm structures form via “differential adhesion, on top of a more general adhesion of cells to each other” is, actually, precisely an inherent aspect of collective cell migration as summarized above for the ovarian border collective.

In our resubmission we will adjust text citing the existing literature to better put into context the reasoning for why PSC formation based on our data is an example of collective cell migration.

(3) That brings up the mechanistic centerpiece of this story, the Slit/Robo system. First: I suggest adding more detailed data from the study by Morin-Poulard et al 2016, in the Introduction, since these authors had already implicated Slit-Robo in PSC function and offered a concrete molecular mechanism: "vascular cells produce Slit that activates Robo receptors in the PSC. Robo activation controls proliferation and clustering of PSC cells by regulating Myc, and small GTPase and DE-cadherin activity, respectively". As stated in the Discussion: the mechanism of Slit/Robo action on the PSC in the embryo is likely different, since DE-cadherin is not expressed in the embryonic PSC; however, it maybe not be THAT different: it could also act on adhesion between PSC cells themselves and their neighbors. What are other adhesion proteins that appear in the late lateral mesodermal structures? Could DN-cadherin or Fasciclins be involved?

We agree with the Reviewer that Slit-Robo signaling likely acts in part on the PSC by affecting PSC cell adhesion to each other and/or to CBs (lines 428-435). As stated in the Discussion, we do not observe Fasciclin III expression in the PSC until late stages when the PSC has already been positioned, suggesting that Fasciclin III is not an active player in PSC formation. Assessing whether the PSC expresses any other of the suite of potential cell adhesion molecules such as DN-Cadherin or other Fasciclins, and then study their potential involvement in the Slit-Robo pathway in PSC cells, would be part of a follow-up study.