Ubx-Collier signaling cascade maintains blood progenitors in the posterior lobes of the Drosophila larval lymph gland

Drosophila larval hematopoiesis occurs in a specialized multi-lobed organ called the lymph gland. Extensive characterization of the organ has provided mechanistic insights into events related to developmental hematopoiesis. Spanning from the thoracic to the abdominal segment of the larvae, this organ comprises a pair of primary, secondary, and tertiary lobes. Much of our understanding arises from the studies on the primary lobe, while the secondary and tertiary lobes have remained mostly unexplored. Previous studies have inferred that these lobes are composed of progenitors that differentiate during pupation; however, the mechanistic basis of this extended progenitor state remains unclear. This study shows that posterior lobe progenitors are maintained by a local signaling center defined by Ubx and Collier in the tertiary lobe. This Ubx zone in the tertiary lobe shares several markers with the niche of the primary lobe. Ubx domain regulates the homeostasis of the posterior lobe progenitors in normal development and an immune-challenged scenario. Our study establishes the lymph gland as a model to tease out how the progenitors interface with the dual niches within an organ during development and disorders.

We disagree with this point and would like to draw the reviewer's attention to the fact that the progenitors in the posterior lobes were largely uncharacterized. Most of the studies referred to them as a homogeneous pool of progenitors.
Based on our expression studies, the results in the first panel illustrate that heterogeneity exists in the posterior lobes. We, therefore, have attempted to update the existing model (summarized in Sorry for the confusion. We understand the issue raised. However, all of our analyses reproducible indicated the existence of two domains. The artefact is sample-specific, created most likely by the twisting of the slender lobes while mounting. To remove the confusion, we have replaced it with a representative image in the revised version. Thanks for pointing this out. 7. Figure 1 title: Please rephrase "Posterior Lobes are a heterogeneous bunch of cells" to academic language.
We have rephrased it with the following statement: "Posterior lobes house heterogeneous cell populations" Please refer to Page No: 34 and Line No : 842. 8. In Figure 2, please explain how the arrows are drawn to define the secondary vs. tertiary lobe.
As accepted in the field, our reference point is the pericardial cells (indicated by coloured arrows) intercalated between the lymph gland lobes. We understand that the description is needed for all figure legend and have included that in the revised version. 9. In Figure S2, I find the choice of staging to 110h AEH incompatible with the staging in Figure   1, if the authors want to show that Ance-region previously describe in Figure 1 is Ubx+, use the same staging is essential.
As mentioned in the text, regression of Collier and Ubx though initiates early on but is most evident during 110h AEH. In the absence of markers that are limited to one vs the cell type, we felt the best stage to illustrate the regression phenomena is 110 AEH.
10. Figure 4A-J, Ubx and Pxn in the same channel is very difficult for readers to visualize in the Since antibodies for Ubx and Pxn are raised in mice, we had faced this technical difficulty.
However, we could interpret the data since Ubx is nuclear and Pxn is membranous. Moreover, in the current version, we have also analyzed loss of Ubx with Ubx(M1)-Gal4 and observed similar results. Please refer to Figure 5 of the revised manuscript.
11. Define how differentiation index is calculated.
Thanks for raising this issue. We have provided the details of our steps in calculating the Differentiation Index in the Material and Methods section of the revised manuscript. Please refer to page No: 24 and Lines: 574-585.
12. I don't understand what is sar1 and why downregulating it blocks niche signaling. Moreover, the result in Figure S4 seems important enough to be incorporated to Figure 4.
Newly synthesized proteins are transported from the endoplasmic reticulum to Golgi via coat protein complex II (COPII), the formation of which is regulated by Sar1. Thus, the knockdown of sar1 leads to the intracellular accumulation of proteins that are otherwise meant to be released extracellularly. Please note that downregulation of sar1 has been previously employed in the lymph gland to block cell-cell communication (Destalminil-Letourneau, M., Morin-Poulard, I., Tian, Y., Vanzo, N. and Crozatier, M., 2021. The vascular niche controls Drosophila hematopoiesis via fibroblast growth factor signaling. Elife, 10, p.e64672).
We have now included a short description in the text. Please refer to Page No:14 and Lines. 323-330.
Following reference on the function of Sar1 and its use in blocking cell-cell communication has also been incorporated. 12. What is Figure 4K and L? There is no explanation in the article.
We want to draw your attention once again. It has been called and explained in the result section.
We have elaborated on it in the revised version. Please refer to Page No: 13 and lines: 297-300.
13. Figure 5, what is the timing of this experiment? I find overexpression of Ubx in a domain where its expression is already high somewhat puzzling. For the experiment mentioned, we speculated was that Ubx over-expression from the Ubx domain could strengthen the niche functionality by boosting the maintenance signal, which then can help in rescuing the ectopic differentiation due to wasp parasitism. In the revised version of our manuscript, we have demonstrated that Hh levels (transcriptional reporter: hhF4fGFP) increase upon Ubx over-expression, reflecting the increase in niche functionality. Please refer to Line 97 "unique zonation signifying distinct cell types within these lobes" please rephrase for clarity.
We have rephrased this to "unique pattern of zonation signifying distinct cell types within these lobes" Please refer Page: 5 and Line: 109 of the revised manuscript. Thank you very much for your appreciation. It means a lot to us.
The presented data convincingly allow to distinguish the progenitor status of the secondary and tertiary lobes and draw some new conclusions on the role of homeotic genes in controlling hematopoiesis in the Drosophila LG. They also expand the LG niche concept by drawing parallels between Col/Antp-expressing cells in primary lobes and Col/Ubx cells in secondary lobes. Altogether, this ms should represent a valuable contribution to our knowledge of Drosophila hematopoiesis. Drosophila is a reference genetic model for studying animal innate immunity and this paper should be of interest to a wide audience.
Thanks a lot. We are much encouraged by your valuable comments. Deeply appreciate your time and input in reviewing our manuscript.
However, before publication, important revisions are needed.
The conclusions of the authors are essentially based on expression/RNAi patterns obtained with one Gal4 driver. The present data need to be completed by a full description of this driver activity, compared to Col/Kn Antibody/ISH stainings. Morover, the author's conclusions require to be supported by additional experiments, using a driver not expressed in PSC cells (the « classical » niche).
Thanks for your guidance. To address your concerns, we have done the following experiments: A. The present data need to be completed by a full description of this driver activity, compared to Col/Kn Antibody/ISH stainings.
The Kn/Col-Gal4 driver lines were previously described (Krzemien et al., 2007, Tokusumi et al., 2017, Carayon et al., 2020. However, we understand the concern and have done antibody labeling of Col to demonstrate the expression domains of the two Gal4 constructs used for our analysis.
Please refer to S2 Figure  Ideas of the authors on the potential role of Col/Ubx "progenitor" cells traced in adults also need to be clarified, and a summary figure added. This is an mportant point, since, no functional data support the existence of adult hematopoiesis in Drosophila, notwithstanding the fact that the persistence of some hematopoietic progenitors in adults is itself subject to caution.
We have performed lineage tracing analysis with a posterior lobe specific Gal4 in the revised manuscript. Ubx(M1)-Gal4 on lineage tracing throughout development marks the posterior lobes.
However, the live expression is limited to the anterior part of the tertiary lobe/PLSC co-localizing with Ubx antibody (please refer to the S5 Figure). We have summarized our previous and the recent result in Figure 7 of the revised manuscript.
Studies employing PH3 and BrdU have shown that hemocytes in the hematopoietic hub have the potential to proliferate upon bacterial challenges (Ghosh et al., 2015, Ghosh et al., 2018. This simplification is triply misleading. 1) Formally, there is no valuable reason to call this driver Knot/kn-Gal4, since it is not an insertion in the locus. There are many drivers made from col/kn introns or upstream/downstream regions which have already been characterised. This one, GMR13A11. col/kn GMR lines has already been studied by other labs (Tokosumi et al ;2017 ;Carayon et al., 2020), showing that GMR13A11 only reproduces part of the col :kn expression in the LG.
We agree with the reviewer; these papers indicate that GMR13A11 (Kn-Gal4) partially reproduces Col/Kn expression in LG. We chose Kn-Gal4 as we found it to be very specific for its expression in LG. Least expression was found in other tissues in comparison to pCol-Gal4.
Moreover, the Kn-Gal4 and pCol-Gal4 time series with Col antibody in the recent version of this manuscript further endorses our decision to use Kn-Gal4, which seems to co-localize with the high Col antibody cells, which we wanted to address in our study. Please refer to the S2 Figure in 3) Unlike Col/Kn, GMR 13A11 is not expressed in primary lobe progenitors but strongly expressed in the PSC. A key conclusion of the paper, namely that the anterior tertiary lobe acts in a non cell-autonomous manner (as a niche) for maintaining secondary lobe progenitors, is based on one experiment using a similar driver (pCol-Gal4/Ubx RNAi experiment; see also comments on Fig. 4). This conclusion needs to be confirmed using a driver not expressed in the PSC. In other terms, that the PSC does not act as a niche for secondary lobe progenitors needs to be ruled out. What is crucially missing is a confirmation of the author's conclusions by repeating experiments using a driver expressed in posterior lobes but NOT EXPRESSED in PSC cells, unlike GMR13A11 or Pcol85.
As mentioned earlier, UbxM1-Gal4 only expresses in the posterior lobes and is totally absent in the primary lobe (S5 Figure). Using this Gal4, we have carried out the functional assays to address the reviewer's concern.
In order to assess the role of primary lobe PSC in the maintenance of posterior lobe progenitors, we knocked down hh and sar1 from the PSC and assayed differentiation in the posterior lobes.
Intriguingly, we do see a few cells differentiating in the posterior lobes (please see Figure below).
Based on this observation, we can infer that the primary lobe PSC does have a partial contribution to the maintenance of posterior lobes.

Fig 4, 5 and S5: What is crucially missing is a confirmation of the author's conclusions by
repeating experiments using a driver expressed in posterior lobes but NOT EXPRESSED in PSC cells, unlike GMR13A11 or Pcol85.
Please refer to our response above. We have repeated all the experiments with posterior lobe specific driver, which is not expressed in the PSC. However due genetic constraint, we were unable to repeat the experiment shown in S5 (which is S11 of the current manuscript). To perform this experiment with posterior lobe specific driver :Ubx(M1)-Gal4 which is on third chromosome was technically difficult, since both D4-Lacz and UAS-Ubx are also on third chromosme. Except this one experiment rest have beenrepeated with the posterior lobe specific driver.   Following the reviewers' suggestion, we have used a posterior lobe-specific driver Ubx(M1)-Gal4 for this experiment. Please refer to S7 Figure. As suggested by the reviewer, Ubx levels were assessed upon wasp infection. We employed Pxn-YFP so that we can visualize the differentiation status upon wasp parasitism. A significant drop in the levels of Ubx in posterior lobes was evident upon immune challenge (S8 Figure).  Results.

All figures
Indicating Pxn, Kn/Col, Dad etc, in red on the figure panels is misleading. What is detected is Pxn-YFP and GMR13A11>mCD8GFP, etc, expression, not the endogenous protein expression.
For other reporters, the labelling is correct. Please chek all figures for consistency.
Thanks for pointing it out. All the changes have been made in the current version of the manuscript.

Fig2S
. What is the colPS mutation which is not listed among fly stocks? Is it col1 ? In this case please refer to is as col1.
Thanks for drawing our attention. Yes, it is col 1 (Genotype: w*; kn col-1 / CyO; P{col5-cDNA.C}/TM6B). We have made these changes in the current version. This is due to excessive differentiation (as observed by the P1 ) in col loss of function. To represent the variation often encounter in the otherwise highly differentiated lymph glands of col 1 , we have included two examples in the same panel (S3 F-G' Fig). In G, the tertiary lobe is drawn without evidence from DAPI staining ? Sorry for the confusion created. We have re-processed the same image to clear the confusion Fig. S3 The level of Ser expression in tertiary lobes seems more similar to Ser expression in primary lobes outside the PSC in early third instar (Crozatier et al., 2004 ;Spratford et al., BioRxiv, 2020) than in the PSC. Unlike frc, Ser and Lamin-C are expressed in the entire tertiary lobes, and not restricted to the posterior domain.
As mentioned in the text, at 96hr AEH the expression of these markers include a subset of Ubx positive cells.   (please refer to S11 I, S11 L Figs) Reviewer #3: The lymph gland of Drosophila plays a significant regulatory role in larval hematopoiesis. Several functional cellular compartments and regulatory factors have been identified, however the studies focused mainly on the anterior lobes of the organ. Developmental studies suggested that the posterior lobes and their regulation is different from those of the primary lobes, but their regulation, their role in hematopoiesis and in the immune response has not been explored. The Authors of the presented studies reveal the functional heterogeneity of hematopoietic progenitors residing in these lobes and identify regulatory factors acting in the separate niches and responsible for the regulation of the distinct functional progenitor populations.
Thanks a lot for your time and comments. They are deeply appreciated.

Minor comments:
Lines 65 and 691: SHRESTHA and GATEFF are in capital