Src42A is required for E-cadherin dynamics at cell junctions during Drosophila axis elongation

  1. Lenin Chandran
  2. Wilko Backer
  3. Raphael Schleutker
  4. Deqing Kong
  5. Seyed A. H. Beati
  6. Stefan Luschnig
  7. H.-Arno J. Müller

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Abstract

Src kinases are important regulators of cell adhesion. Here, we have explored the function of Src42A in junction remodelling during Drosophila gastrulation. Src42A is required for tyrosine phosphorylation at bicellular (bAJ) and tricellular (tAJ) junctions in germband cells, and localizes to hotspots of mechanical tension. The role of Src42A was investigated using maternal RNAi and CRISPR-Cas9-induced germline mosaics. We find that, during cell intercalations, Src42A is required for the contraction of junctions at anterior-posterior cell interfaces. The planar polarity of E-cadherin is compromised and E-cadherin accumulates at tricellular junctions after Src42A knockdown. Furthermore, we show that Src42A acts in concert with Abl kinase, which has also been implicated in cell intercalations. Our data suggest that Src42A is involved in two related processes: in addition to establishing tension generated by the planar polarity of MyoII, it may also act as a signalling factor at tAJs to control E-cadherin residence time.

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    Reply to the reviewers

    General response to the reviewer

    We thank all reviewers for their constructive comments on our manuscript. We were very pleased to see that the reviewers found our study ‘…represent new insight in the field’ (rev#1) and ‘…contains important and exciting novel findings’ (rev#2), and ‘…gives a more detailed perspective on how Src proteins (Src42A in Drosophila) control epithelial stability and the contraction of specific surfaces of epithelial cells’ (rev#3). The reviewers raised a number of specific points that we partially addressed already in a preliminary revision of the manuscript. Some more points will require some additional experiments that we will incorporate in a fully revised version of the manuscript.

    __Reviewer #1 __

    __(Evidence, reproducibility and clarity (Required)): __ Highest priority: *1) The Src42A knockdown and germline clone experiments both cause defects in cellularization (Fig. 2B and 9A), which could result in differences in the state of the blastoderm epithelium (cell size, cell number, structural integrity, organization, etc.) between the experimental and control conditions. In addition, Src42A knockdown appears to affect the size and shape of the egg (Fig. 9A and 9C). The manuscript would be strengthened if the authors included data to demonstrate that the initial structure of the epithelium is mostly normal (quantifications of cell size, number, etc.) in the Src42A RNAi condition, as this would bolster the argument that *germband extension, rather than due to indirect effects resulting from the cellularization defects. The authors may have relevant data to do this on-hand, for example using data associated with figures 1, 3, 6, and 9.

    Response:

    The cellularization phenotype of src42A knockdown embryos has a penetrance of about 50% and exhibits a variable expressivity. We attempted to characterize this phenotype in detail, but failed to identify any dramatic differences in cellularization of the src42A knockdown embryos compared to wild type. The localization of E-cadherin, in turn is not affected, but occasionally, nuclei are dropping out of the blastoderm before cellularization is accomplished. This can result in patches of irregular cellularization, but the blastoderm epithelium in stage 6 embryos did not display major defects in overall structure. We will present additional data on the cellularization phenotypes in the fully revised manuscript. As the referee suggested, we will analyze our data to determine potential effects on the cell size, cell number and overall organization of the blastoderm before germband extension. We plan to present these data as an additional Suppl. Mat. Figure in the full revision.

    Lower priority:

    1. Figure 8 - in my opinion, using a FRAP or photoconversion approach would be a more convincing demonstration of differences in E-cadherin residency times / turnover rate than time-lapse imaging of E-cadherin:GFP alone. Authors should decide whether this improvement is worth the investment.

    Response:

    We thank the reviewer for this comment. While we believe that the data presented in Fig. 8 demonstrates a significant difference in the E-cadherin residence time based on E-cadherin-GFP fluorescence intensity, we agree with the referee that FRAP analyses would provide additional evidence to support our conclusion. For the full revision, we will therefore attempt to perform FRAP-experiments on src42A knockdown embryos expressing E-cadherin-GFP and compare the recovery time to the wild type.

    Reviewer #1 (Significance (Required)):

    The manuscript by Backer et al. examines the function of Src42A in germband extension during Drosophila gastrulation. Prior studies in the field have shown that Src family kinases play an important role in the early embryo, including cellularization (Thomas and Wieschaus 2004), anterior midgut differentiation (Desprat et al. 2008), and germband extension (Sun et al. 2017; Tamada et al. 2021). In this study, the authors showed that Src42A was enriched at adherens junctions and was moderately enriched along junctions with myosin-II. They then showed that maternal Src42A depletion exhibits phenotypes, starting with cellularization and including a defect in germband extension. The authors focus on defects in germband extension and found that Src42A was required for timely rearrangement of junctions and that the Src42A RNAi phenotype is enhanced by Abl RNAi. Finally the authors show that E-cadherin turnover is affect by Src42A depletion.

    Overall, this study provided a higher resolution description of how Src42A regulates the behavior of junctions during germband extension. I thought the authors conclusions were well supported by the data and represent new insight in the field.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    Summary: Chandran et al. investigate the role of Src42A in axis elongation during Drosophila gastrulation. Using maternal RNAi and CRISPR/Cas9-induced germline mosaics, they revealed that Src42A is required to contract junctions at anterior/posterior cell interfaces during cell intercalations. Using time-lapse imaging and image analysis, they further revealed the role of Src42A in E-Cad dynamics at cell junctions during this process.

    By analyzing double knockdown embryos for Src42A and Abl, they further showed that Src42A might act in parallel to Abl kinase in regulating cell intercalations. The authors proposed that Src42A is involved in two processes, one affecting tension generated by myosin II and the other acting as a signaling factor at tricellular junctions in controlling E-Cad residence time. Overall, the data are clear and nicely quantified. However, some data do not convincingly support the conclusion, and statistical analyses are missing for an experiment or two. Methods for several quantifications also need improvement in writing. Also, several figures (Figures 6-8) do not match the citation in the text and need to be corrected.

    Page and line numbers were not indicated in the manuscript. For my comments, I numbered pages starting from the title page (Title, page 1; Abstract, page 2, Introduction, pages 3-6; Results, pages 7-14; Discussion, pages 15-18; M&M, 19-23; Figure legends, 28-30) and restarted line numbers for each page. For Figures 6-8 that do not match the citation in the text, I still managed to look at the potentially right panels. All the figure numbers I mention here are as cited in the text. My detailed comments are listed below.

    Response:

    We apologize for the lack of organization of the manuscript and the figure numbering. In the revised version we have added page numbers, line numbers and we corrected the figure numbers.

    Major comments:

    1. b-Cat/E-Cad signals at the D/V and A/P junctions in Src42Ai (Figs. 5-6). These data are critical for their major conclusion and should be demonstrated more convincingly.

    In Fig. 5A, the authors said, "When the AP border was cut, the detached tAJs moved slower in Src42Ai embryos compared to control (Fig. 5A)". However, even control tAJs do not seem to move that much in the top panels, and I found the images not very convincing.

    Response:

    We thank the referee for commenting on the lack of clarity in the presentation of the data. The overall movement within the first 10 seconds after the laser cut (determined by movement of adjacent D/V tAJs from each other) was about 2 µm in the wildtype, while in the mutant it was 1 µm. Despite this 50% difference, it may be difficult to appreciate this difference from looking at Fig. 5A in our original submission. The yellow lines in Fig 5A only showed the region of the cut, but did not indicate the movement of the tAJ from each other, which may have led to a distraction from the actual movement. We will change the annotation and the marks within the figure to visualize the movement much more clearly in the full revision. In the fully revised manuscript, we will also add movies from the experiments including marks of the tricellular junctions to follow the displacement as part of the Supplemental Material.

    Based on the genetic interaction between Src42A and Abl using RNAi (Fig. 7), the authors argue that Src42A and Abl may act in parallel. However, the efficiency of Abl RNAi has not been tested. It can be done by RT-PCR or Abl antibody staining. Also, the effect of Abl RNAi alone on germband extension should be tested and compared with Src42A & Abl double RNAi embryos. I expect the experiments can be done within a few weeks without difficulty.

    Response:

    We agree with the referee that it is important to determine the level of depletion in Abl RNAi embryos in order to interpret the genetic relationship between Abl and Src42A. In the full revision of the manuscript, we will follow the advice of the referee and analyze the knockdown, preferably by antibody labeling with an anti-Abl antibody. We will also generate single knockdowns of abl in embryos and determine their effect on germband extension compared to wildtype and src42/abl double knockdown.

    Minor comments:

    Fig. 2

    • Fig. 2B: Higher magnification images of the defective cytoplasm can be shown as insets.

    Response:

    We will add some higher magnification images of the cellularization phenotype in the full revision of the manuscript. In addition, as mentioned in the response to reviewer #1, we will provide a more detailed analysis of the cellularization in src42Ai embryos in the fully revised manuscript.

    • Fig. 2E: A simple quantification of the penetrance of cuticle defects in Src42A mutants and RNAi will be helpful, as shown in Fig. S3.

    Response:

    In the full revision, we will add the quantification of the occurrence of the different classes of cuticle phenotypes.

    Fig. 9

    • Fig. 9A: Magnified views of the cytoplasmic clearing can be added as insets.

    Response: As described in our response to the comments made by referee #1, we will add a more detailed analysis of the cellularization phenotype in the full revision.

    Page 14, lines 9-10: More explicit description of the phenotype rather than just "stronger compared to Src42Ai" will be helpful.

    Response:

    In the full revision, we will add a more detailed description of the phenotype and re-analyze and present data on the hatching rate, stage of lethality and cuticle phenotypes.

    __Reviewer #2 (Significance (Required)): __ This work revealed the role of Src42A in regulating germband extension. A previous study suggested the roles of Src42A and Src64 in this developmental process using a partial loss of both proteins (Tamada et al., 2021). Using different approaches, the authors demonstrated a role of Src42A in regulating E-Cad dynamic at cell junctions during Drosophila axis elongation. Most of the analyses were done with maternal knockdown using RNAi, but they successfully generated germline clones for the first time and confirmed the RNAi phenotypes. Overall, this work contains important and exciting novel findings. This work will be of general interest to cell and developmental biologists, particularly researchers studying epithelial morphogenesis and junctional dynamics. I have expertise in Drosophila genetics, epithelial morphogenesis, imaging, and quantitative image analysis.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    Chandran et al. report on the function of Src42A during cell intercalation in the early Drosophila gastrula. They create a Src42A-specific antibody (there are two Src genes in the fly genome) and examine the localization of Src42A and observe a planar-polarized distribution at cell interfaces. They then measure cell-contractile dynamics and show that T1 contraction is slower after Src42A disruption. The authors then argue that Src42A functions in a parallel pathway to the Abl protein, and that E-cadherin dynamics (turnover) is altered in Src42A disrupted embryos. Src function at these stages has been studied previously (though not to the degree that this study does), and in some respects the manuscript feels a little preliminary (please label figures with figure number!), but after editing this should be a polished study that merits publication in a developmentally-focused journal.

    1. Does the argument that Src42A has two functions fully make sense? Myosin II function is known to affect E-cadherin stability (and vice versa), so it seems that Src42A could affect both MyoII and Ecad by either decreasing Myosin II function/engagement at junctions or by destabilizing Ecad.

    Response:

    We thank the referee for raising an important point that we may not have discussed appropriately in our initial submission. We agree that the reciprocal relationship between actomyosin and E-cadherin might not be reflected equivocally in our manuscript. As the referee points out, Src42A could affect both MyoII planar localization and E-cadherin dynamics through the same pathway. Previous studies showed that Src is involved in translating the planar polarized distribution of the Toll-2 receptor by recruiting Pi3-Kinase activity to the Toll-2 receptor complex resulting in planar polarized distribution of MyoII at the A/P interfaces. These data, however do not address the possibility that a well-known Src target, the E-cadherin/ß-Catenin complex, which is extensively remodeled in germband extension contributes to the delay in germband extension. The observed defects in both studies can be attributed to both a defect in abnormal planar polarization of MyoII and the abnormal dynamics of the E-cadherin/ß-catenin complex. In either of these cases, we suggest that Src42A phosphorylates distinct substrates, the Toll-2 intracellular domain in the MyoII planar polarity pathway and the E-cad/ß-Cat complex controlling E-cad dynamics. Given the relationship between MyoII and E-cadherin, however, it is not possible to decide whether these two effects are independent functions of Src42A or are consequences of each other. Since we cannot resolve a possible epistatic relationship between these potential two activities of Src42A, we decided to extend the discussion on this topic by taking both possible scenarios into account and discussing them appropriately. We will add this discussion in the full revision of the manuscript.

    ) One obvious question that arises is the nature of cleavage defects that are mentioned that happen previously to intercalation. For example, is E-cad normal prior to intercalation initiating? How specific are the observed defects to GBE?

    Response:

    please see response to referee #1

    1. Pg. 10, "the shrinking junction along the AP axis strongly reduces its length with an average of 1.25 minute" - what is this measurement? How much is "strongly"?

    Response:

    We thank the referee for pointing out our inappropriate qualitative statement of the experimental data, which was indeed misleading. The measurement of the shrinking junction was based upon the time it takes for the AP interface junction between two adjacent vertices on the DV axis to shrink into a single 4-cell vertex. The time for this contraction was on average 1 minute 25 seconds. The data in Fig.4 A’,C show that after 2 minutes in the control embryo 100% of the observed AP junctions have collapsed and the extension of the new DV junction along AP axis has begun. At the same timepoint of 2 minutes in the src42A knockdown, we show in Fig. 4B’,D that the shrinking of the AP junction interface has still not been completed in 60% of the cases.

    In the full revision, we will remove the qualitative statement and replace it with a correct description of the measurements taken and will refer to the data described in Fig. 4 A-D.

    1. Also pg. 10, "the AP junction was not markedly reduced after 1 minute" - what is the criteria for this statement? X%? 1 minute is very specific, it feels like how much of a reduction/non-reduction should also be specific.

    Response:

    please see response to point 3.

    Reviewer #3 (Significance (Required)):

    This study gives a more detailed perspective on how Src proteins (Src42A in Drosophila) control epithelial stability and the contraction of specific surfaces of epithelial cells.

    Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #2 and #3 noted that the manuscript was somewhat unorganized with regard to lacking the numbering of pages, lines and figures. We also noted that in the submission process the figures were not presented in the correct order. In the preliminary revision of the manuscript, we fixed these problems to facilitate the evaluation of our transferred manuscript by editorial boards.

    In addition, we also addressed issues that the referees mentioned by editing the text according to their comments. We also addressed problems regarding the presentation of the figures and statistical analyses of the data. The following changes were made:

    1. We added page numbers and line numbers.
    2. We added figure numbers to the figure panels.
    3. We corrected ordering of figures in the transferred manuscript.
    4. We addressed the following comments by statistical analyses, editing the text and the figures:

    Regarding comments from Reviewer #1:

    Highest Priority:

    1. There is a discrepancy in the staging of embryos used between some of the analyses, which make it hard to interpret some of the data. For example, characterization of the knockdowns in Fig. 1A and B are based on stages 10 and 15, whereas the majority of the paper is focused on earlier stages 6 - 8 during germband extension (e.g., Fig. 1D). The analysis for Fig. 1B would be more meaningful if it was done on the same stages used for subsequent phenotypic analysis so they can be directly compared.

    Response:

    We thank the referee for pointing out an apparent misunderstanding caused by the description of Fig. 1A,B. The data presented in Fig.1A and 1B do not show RNAi knockdown experiments, but show a comparison between embryos that are heterozygous or homozygous for the loss-of-function allele src42A26-1. These data were intended to demonstrate that zygotic mutants still maintain levels of maternal Src42A protein up until late stages of development. Data for embryos at an earlier stage (stage 5) were shown in the Supplementary Fig. S1E, where no difference in protein levels of Src42A can be observed between heterozygous and homozygous zygotic src42A26-1 embryos.

    At the beginning of the results sections 1 and 2 of the preliminary revised manuscript, we added a sentence to address the referee’s concern that earlier stages exhibit no difference in protein levels and will refer to Fig. S1E. We also more explicitly spelled that out that the experiment (referring to Fig.1A,B and S1) was intended to look at zygotic mutants and to demonstrate that our novel Src42A antibody was able to detect the reduction of maternal Src42A protein in mid- to late-stage homozygous zygotic embryos.

    1. There is incongruence between figures in terms of which junctional pools (bAJs vs. tAJs) of beta-catenin and E-cadherin are quantified that makes it difficult to draw comparisons between analyses. For example, pTyr levels are examined for both bAJs and tAJs in Figure 3, however, only tAJs are considered in Fig. 8. Similarly, in some cases planar cell polarity is considered (e.g., comparison of levels at AP vs DV bAJs in Fig. 6 and 9), and in other cases (e.g. Fig. 8) it is not.

    Response:

    We thank the referee for commenting on the different readouts for different pools of cell junctions in our experiments. In our study we considered effects on src42A on both, bAJs and tAJs by RNAi knockdown of src42A. We decided to present the data for bAJ and tAJ in separate figures for clarity and structure. For example, the data for the effect of src42A knockdown on the planar polarized distribution on bAJs of E-cadherin were presented in Fig.6, while the effect on E-cadherin residence time in tAJs were presented in Fig.8. The analysis pTyr levels considered both pools in order to determine whether src42A knockdown leads to an overall reduction of pTyr levels or to a reduction in a specific junctional pool. From our data we conclude that pTyr levels show a similar reduction in both, the bAJ and the tAJ junctions.

    In order to address the reviewer’s comment, we have linked the figures more stringently with the results text of the preliminary revision. We only referred to the reduction in PTyr levels in Fig. 3 to point out that both junctional pools are affected by reduced PTyr in src42i embryos. Furthermore, we referred to the individual figure panels when addressing junctional pools and explain the rationale to focus on particular pools (bAJs or tAJ) in the experiments in detail. For Fig. 6 we point out in the preliminary revised manuscript that we focus the analyses on the known planar polarized distribution of beta-catenin and E-Cadherin.

    Lower priority:

    1. Introduction, 2nd paragraph - The modes of cell behaviors described to drive cell intercalation leaves out another clear example in the literature - Sun et al., 2017 - which describes a basolateral cell protrusion-based mechanism. While the authors cite this paper later, leaving it out when summarizing the state of the field misrepresents the current knowledge of the range of mechanisms responsible.

    Response:

    We thank the referee for this remark. In the preliminary revision, we have added to the introduction that the cell behaviors associated with germband elongation include apical and basolateral rearrangements of the cells indicating that basolateral protrusions also contribute to the set of mechanisms that drive germ band elongation.

    1. 'defective cytoplasm' - this term is confusing, and could perhaps be replaced with 'cellularization defect', or something similar.

    Response:

    We agree that the term we applied for the cellularization defect may be misleading. The observation, we intended to describe with the term was a defect in the cytoplasmic clearing which occurs in the last syncytial division and the beginning of the cell formation process. We changed the description of this observation according now refer to the defect in the preliminary revised manuscript as ‘cytoplasmic clearing defect’.

    1. Tests of statistical significance are not uniformly applied across the figures. For instance, Figures 3G + H indicate statistical significance, but Fig. 3D + E do not. Performing statistical tests throughout the paper, or clearly articulating a rationale when they are not used, would strengthen the manuscript. Specifically, the authors should consider this for Fig. 3D + E, and Fig. 7D + E, to support their arguments that rates of germband extension are different between conditions.

    Response:

    We agree with the reviewer and have provided statistical analysis for the data displayed in Fig. 3D,E and Fig. 7D,E in the preliminary revision of the manuscript.

    1. Page 12 - "We found that Src42A showed a distinct localization at the tAJs (Fig. 1B)": Figure 1B shows a quantification of levels at bAJs, not tAJs.

    Response:

    In the preliminary version of the revised manuscript, we added a quantification of the localization of Src42A at the tAJs as a part of Suppl Fig. S4. In Fig. S4A-C we show that Src42A is enriched in comparison to the bAJs.

    Regarding comments from reviewer #2:

    Major Comments:

    In Fig. 6A, b-Cat signals look fuzzier and dispersed and have more background signals in the control, compared to the Src42Ai background. Also, b-Cat signals in the control image do not seem to show enrichment at the D/V border, as shown in Tamada et al., 2012.

    Response:

    We agree with the referee that the image in Fig. 6A for the control is fuzzier and looks dispersed. This is due to the fixation method that we used. In this experiment we did not apply heat fixation, but used formaldehyde fixation in which b-catenin protein, in addition to the junctional pool, is also maintained in the cytoplasm creating the fuzzy cytoplasmic staining. We chose to do this in order to be able to co-immunolabel the embryos with b-catenin and E-cadherin antibodies; the latter staining is not working with the heat fixation applied in the Tamada et al. 2012 paper. Despite the slightly lower quality of the staining, the quantification of the data clearly indicated an effect of src42A knockdown on the planar polarized distribution of E-cad/b-cat complex does show an enrichment. In the preliminary revision added a note to the figure legend to indicate the fact that the fixation procedure was not optimized for b-catenin junctional staining. In the preliminary revision we also added a quantification of live imaging data recording E-cadherin-GFP in wild-type and src42Ai embryos. We present these additional data in Fig. S5 in the preliminary revision of the manuscript. These data are consistent with the results in Fig. 6 from the immunolabeling and support our conclusion that E-cadherin AP/DV ratio is increased in Src42A knockdown embryos.

    In Fig. 6B, C, it is not clear how the intensity was measured and how normalization was done. Was the same method used for these quantifications as "Protein levels at bicellular and tricellular AJs" on pages 21-22? Methods should be written more explicitly with enough details.

    Response:

    We thank the referee for pointing out the lack of detail in explaining how the quantification was done. In the preliminary revision of the manuscript, we extended a paragraph entitled ‘Protein levels at bicellular and tricellular junctions’ in the methods section that will serve this purpose and describe the methods that were applied for each quantification and the method as to how the data were normalized.

    Does each sample (experimental repeat) for the D/V border in Fig. 6B match the one right below for the A/P border in Fig. 6C? It should be clearly mentioned in the figure legend. The ratio of the DV intensity to AP intensity will better show the compromised planar polarity of the b-Cat/E-Cad complex.

    Response:

    We thank the reviewer for pointing out a lack of clarity in our presentation. The experimental repeats for each measurement do indeed match, i.e. the measurement of the DV border matches the same adjacent 4-cell pair in the same embryo and in total 5 distinct embryos were analyzed for each experiment. In the preliminary revision of the manuscript, we explain this detail of the experimental design in the figure legend. In the preliminary revision, we also determined the ratios of DV/AP cell interfaces for b-Cat and E-Cad and added this quantification as panel 6C and 6E for a clearer presentation of the data.

    Minor notes: Page 4, missing comma after "For example"

    Response: The text was edited accordingly.

    Page 4, "inevitable" does not make sense in this context

    Response: We eliminated ‘inevitable’ and replaced it with ‘critical’ to better indicate the importance of Canoe protein for germband elongation.

    Page 7, lines 6-7 - The localization of Src42A in control should be described in more detail and more clearly here.

    Response: In the preliminary revised manuscript, we extended our description of the distribution of Src42A in more detail pointing out its dynamics and differential distribution at distinct plasma membrane domains.

    Supplemental Fig S1

    • Fig. S1D: Based on the head structure and the segmental grooves, the embryo shown here is close to late stage 13/early stage 14, not stage 15.
    • Fig S1E: It will be helpful if the predicted protein band and non-specific bands are indicated by arrows/arrowheads in the figure.

    Response:

    We thank the referee for their careful observation of the embryonic stage. We agree that the embryo was actually a younger stage. In the preliminary revision, we replaced the images with an example of an older stage. We will also add clear annotations as arrows to clearly mark the specific protein bands in Fig. S1E.

    Page 7, lines 21-22

    • "Src42A was slightly enriched at the AP interface" - To argue that, quantification should be provided.

    Response:

    We thank the referee for pointing out a qualitative statement that we made with regard to the distribution of Src42A at the AP cell interfaces. In the preliminary revision of the manuscript, we present an additional quantification of the imaging data of Src42A immunolabeling. In Figure S4A-C, we now present a quantification of the enrichment of Src42A at the tricellular junctions. In addition, the new Fig. S4D,E shows a quantification of the planar polarized distribution of Src42A at the AP cell interfaces.

    Figure 1

    • Fig. 1B: Src42A levels should be compared between control (Src42A/+) and Src42A/Src42A for each stage. It currently shows a comparison between Src42A/Src42A of stages 10 and 15.

    Response:

    We thank the referee for the comment. As indicated in our response to referee #1, the point of this analysis was to (1) provide evidence for the specificity of our new anti-Src42A antibody and (2) to demonstrate the presence of substantial material contribution of Src42A protein in zygotic mutant. We do not see the advantage to provide a detailed developmental Western-blot analysis, but we provide data in Suppl. Mat Fig S1E showing that the level of Src42A is unimpaired in stage 6 zygotic src42A[26-1] homozygous mutant embryos.

    • Fig. 1B: The figure legend says, "dotted line represents mean value and error bars," but there are no dotted lines shown in the figure. Also, what p-value is for ****? It should be mentioned in the figure legend. It also says Src42A levels were normalized against E-Cad intensity here (stages 10 and 15). They have shown that E-Cad levels are affected in Src42A RNAi during gastrulation (Fig. 6). Is E-Cad not affected in Src42A26-1 zygotic mutants at stages 10 and 15?

    Response:

    We thank the referee for pointing out inaccuracies in the presentation and the description of Fig.1B. In the preliminary revision, we emphasized the marks on the graph and provide p-values throughout. Regarding the E-Cadherin levels: E-cadherin levels were altered in src42A RNAi knockdown embryos, but not in zygotic mutants, even at later developmental stages.

    Page 8, line 14

    • "Embryos expressing TRiP04138 showed reduced hatching rates with variable penetrance and expressivity depending on the maternal Gal4 driver used (Fig. 2B)" - Fig. 2B doesn't seem to be a right citation for this sentence.

    Response:

    We agree with the referee and in the preliminary revised manuscript we corrected the reference to the conclusion drawn from Figure 2A’, which does show the relationship of hatching rate to the various maternal Gal4 drivers.

    • Fig. 2C: It will be helpful to indicate two other non-specific bands in the figure with arrows/arrowheads with a description in the figure legend.

    Response:

    In the preliminary revision, we added an arrow to mark the band specific for Src42A and asterisks to mark unspecific bands in Fig 2C.

    Page 9, line 9

    • This is the first time that the fast and the slow phases of germband extension are mentioned. As these two phases are used to compare the Src42A and Src42A Abl double RNAi phenotypes, they should be introduced and explained better earlier, perhaps in Introduction.

    Response:

    We thank the referee for pointing out that the two phases of germband extension were not introduced. We added a sentence to introduce and define the distinct phases of extension movements in the preliminary revision.

    Fig. 3

    • Fig. 3A: It will be helpful to mark the starting and the ending points of germband elongation with different markers (arrows vs. arrowheads or filled vs. empty arrowheads).

    Response:

    In the preliminary revision, we added distinct markers to indicate the start and endpoints of germband elongation to make this figure easier to read.

    • Fig. 3C figure legend: R2 is wrongly mentioned in Fig. 3D, E. Also, R2 (coefficient of determination) needs to be defined either in the figure legend or Materials & Methods.

    Response:

    We thank the referee for pointing this misleading reference to us. In the preliminary revision we corrected the reference to R2 in Fig,3D,E and will describe the definition of R2 in the figure legend.

    • Fig. 3D, E: statistical analysis is missing.

    Response:

    In the preliminary revision, we included a statistical analysis of the data (see ref #1). We changed the figure to indicate the data sets that were analyzed and added the p-values to the figure legend.

    • Fig. 3G and H should be cited in the text.

    Response:

    In the preliminary revision, we added references to Fig 3G,H in the result section to the annotation of Fig.3F).

    • Fig. 3F: It should be mentioned that the heat map is shown for pY20 signals in the figure legend, with an intensity scale bar in the figure.

    Response:

    In the preliminary revision, we added an intensity scale bar to the figure panel and mentioned the relationship to the PY20 signal.

    Fig. 7A: Arrows can be added to mark the delayed germband extension.

    Response:

    In the preliminary revision, we added arrows to mark the anterior and posterior extent of the germband.

    Fig. 8A: It should be mentioned that the heat map is shown for E-Cad signals in the figure legend, with an intensity scale bar in the figure.

    Response:

    In the preliminary revision, we added an intensity scale to the heat map and mention the relationship to the E-cadherin signal in the figure legend.

    Fig. S3G: An arrowhead can be added to the gel image to indicate the band described in the legend.

    Response:

    In the preliminary revision, we added an arrow to help annotating the Src42A-specific bands on the Western blot.

    • Fig. 9B: Arrow/arrowheads can be added to show the absence of the signals in the nurse cells.

    Response:

    In the preliminary revision, we added markers to help recognizing the reduced signal in the nurse cells and the oocyte.

    • Fig. 9C: Indicate the ending point of the germband extension by arrows.

    Response: In the preliminary revision, we added arrows to mark the anterior and posterior extent of the germband.

    Regarding comments from reviewer #3:

    Minor notes: Page 4, missing comma after "For example"

    Response: The text was edited accordingly.

    Page 4, "inevitable" does not make sense in this context Response:

    In the preliminary revision, we eliminated ‘inevitable’ and replaced it with ‘critical’ to better indicate the importance of Canoe protein for germband elongation.

    Description of analyses that authors prefer not to carry out

    Referee #1 point2 and Referee#2 minor comment figure 1. Both referees suggest that figure 1 AB should include earlier developmental stages according to the stages looked at in the RNAi knockdown experiment.

    Response:

    The referees’ comments are likely based on a misunderstanding. The data that the reviewer are referring to present analyses of the zygotic phenotype of embryos homozygous for the src42A26-1 loss of function allele. They are not related to the maternal RNAi knockdown experiments, but were meant to demonstrate the existence and extent of a maternal pool of Src42A protein, that persists even to late stages in development. The maternal knockdown mutants are analyzed in detail at the appropriate stages in Fig. 2.

    As described in our response above, we don’t feel that a detailed developmental stage Western analysis of wildtype and src42A26-1 embryos would provide significant additional insights. As mentioned in our response above, data for an earlier developmental stage (before germband elongation, as requested by the referees, were provided in Suppl. Fig. S1E.

    Referee #1 Point 6) Figure 8E - showing images of multiple tAJs, rather than z-slices of a single vertex, would better support the claim here, as the assertion is that Src42a levels are different between control and sdk RNAi conditions, and not that it varies in the z-dimension.

    Response:

    The image series of Fig. 8E shows one representative example of multiple tAJs that have been imaged for this experiment (n=6 for wild type and n=10 for sdk RNAi). We think that the presentation of Z-slices for this experiment is important as the protein distribution needs to be considered for a larger area along the apical-lateral cell interface. In addition the quantification of the data for multiple tAJs was presented in Fig. 8F,G as a graph. We would therefore rather not change this figure in the revised manuscript.

    Referee #3 suggests that anti MyoII staining should accompany the analysis of tension measurements in the germband.

    As this analysis has already been performed by Tamada et al. 2021, we decided not to reproduce these data, but rather extend the analysis towards tension measurements, which support the findings by Tamada et al. 2021 on a functional level. We do not see the added value of adding MyoII labeling.

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    Referee #3

    Evidence, reproducibility and clarity

    Chandran et al. report on the function of Src42A during cell intercalation in the early Drosophila gastrula. They create a Src42A-specific antibody (there are two Src genes in the fly genome) and examine the localization of Src42A and observe a planar-polarized distribution at cell interfaces. They then measure cell-contractile dynamics and show that T1 contraction is slower after Src42A disruption. The authors then argue that Src42A functions in a parallel pathway to the Abl protein, and that E-cadherin dynamics (turnover) is altered in Src42A disrupted embryos. Src function at these stages has been studied previously (though not to the degree that this study does), and in some respects the manuscript feels a little preliminary (please label figures with figure number!), but after editing this should be a polished study that merits publication in a developmentally-focused journal.

    1. Does the argument that Src42A has two functions fully make sense? Myosin II function is known to affect E-cadherin stability (and vice versa), so it seems that Src42A could affect both MyoII and Ecad by either decreasing Myosin II function/engagement at junctions or by destabilizing Ecad.
    2. One obvious question that arises is the nature of cleavage defects that are mentioned that happen previously to intercalation. For example, is E-cad normal prior to intercalation initiating? How specific are the observed defects to GBE?
    3. Pg. 10, "the shrinking junction along the AP axis strongly reduces its length with an average of 1.25 minute" - what is this measurement? How much is "strongly"?
    4. Also pg. 10, "the AP junction was not markedly reduced after 1 minute" - what is the criteria for this statement? X%? 1 minute is very specific, it feels like how much of a reduction/non-reduction should also be specific.
    5. It seemed odd to mention altered myosin levels but then skip over a measurement of myosin in favor of an indirect measurement such as interface recoil. Again (point 1), it seems that changes in Myosin II recruitment could cause changes in Ecad turnover.

    Minor notes:

    Page 4, missing comma after "For example"

    Page 4, "inevitable" does not make sense in this context

    Significance

    This study gives a more detailed perspective on how Src proteins (Src42A in Drosophila) control epithelial stability and the contraction of specific surfaces of epithelial cells.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Chandran et al. investigate the role of Src42A in axis elongation during Drosophila gastrulation. Using maternal RNAi and CRISPR/Cas9-induced germline mosaics, they revealed that Src42A is required to contract junctions at anterior/posterior cell interfaces during cell intercalations. Using time-lapse imaging and image analysis, they further revealed the role of Src42A in E-Cad dynamics at cell junctions during this process.

    By analyzing double knockdown embryos for Src42A and Abl, they further showed that Src42A might act in parallel to Abl kinase in regulating cell intercalations. The authors proposed that Src42A is involved in two processes, one affecting tension generated by myosin II and the other acting as a signaling factor at tricellular junctions in controlling E-Cad residence time. Overall, the data are clear and nicely quantified. However, some data do not convincingly support the conclusion, and statistical analyses are missing for an experiment or two. Methods for several quantifications also need improvement in writing. Also, several figures (Figures 6-8) do not match the citation in the text and need to be corrected.

    Page and line numbers were not indicated in the manuscript. For my comments, I numbered pages starting from the title page (Title, page 1; Abstract, page 2, Introduction, pages 3-6; Results, pages 7-14; Discussion, pages 15-18; M&M, 19-23; Figure legends, 28-30) and restarted line numbers for each page. For Figures 6-8 that do not match the citation in the text, I still managed to look at the potentially right panels. All the figure numbers I mention here are as cited in the text. My detailed comments are listed below.

    Major comments:

    1. b-Cat/E-Cad signals at the D/V and A/P junctions in Src42Ai (Figs. 5-6). These data are critical for their major conclusion and should be demonstrated more convincingly.

    In Fig. 5A, the authors said, "When the AP border was cut, the detached tAJs moved slower in Src42Ai embryos compared to control (Fig. 5A)". However, even control tAJs do not seem to move that much in the top panels, and I found the images not very convincing.

    In Fig. 6A, b-Cat signals look fuzzier and dispersed and have more background signals in the control, compared to the Src42Ai background. Also, b-Cat signals in the control image do not seem to show enrichment at the D/V border, as shown in Tamada et al., 2012.

    In Fig. 6B, C, it is not clear how the intensity was measured and how normalization was done. Was the same method used for these quantifications as "Protein levels at bicellular and tricellular AJs" on pages 21-22? Methods should be written more explicitly with enough details.

    Does each sample (experimental repeat) for the D/V border in Fig. 6B match the one right below for the A/P border in Fig. 6C? It should be clearly mentioned in the figure legend. The ratio of the DV intensity to AP intensity will better show the compromised planar polarity of the b-Cat/E-Cad complex.

    1. Based on the genetic interaction between Src42A and Abl using RNAi (Fig. 7), the authors argue that Src42A and Abl may act in parallel. However, the efficiency of Abl RNAi has not been tested. It can be done by RT-PCR or Abl antibody staining. Also, the effect of Abl RNAi alone on germband extension should be tested and compared with Src42A & Abl double RNAi embryos. I expect the experiments can be done within a few weeks without difficulty.

    Minor comments:

    Page 2, line 14 - The abbreviation for tAJs was not introduced before.

    Page 7, line 6 - A reference should be cited for the Src42A26-1 allele.

    Figure 1

    • Fig. 1B: Src42A levels should be compared between control (Src42A/+) and Src42A/Src42A for each stage. It currently shows a comparison between Src42A/Src42A of stages 10 and 15.
    • Fig. 1B: The figure legend says, "dotted line represents mean value and error bars," but there are no dotted lines shown in the figure. Also, what p-value is for ****? It should be mentioned in the figure legend. It also says Src42A levels were normalized against E-Cad intensity here (stages 10 and 15). They have shown that E-Cad levels are affected in Src42A RNAi during gastrulation (Fig. 6). Is E-Cad not affected in Src42A26-1 zygotic mutants at stages 10 and 15?

    Page 7, lines 6-7 - The localization of Src42A in control should be described in more detail and more clearly here.

    Supplemental Fig S1

    • Fig. S1D: Based on the head structure and the segmental grooves, the embryo shown here is close to late stage 13/early stage 14, not stage 15.
    • Fig S1E: It will be helpful if the predicted protein band and non-specific bands are indicated by arrows/arrowheads in the figure.

    Page 7, lines 21-22

    • "Src42A was slightly enriched at the AP interface" - To argue that, quantification should be provided.

    Page 8, line 14

    • "Embryos expressing TRiP04138 showed reduced hatching rates with variable penetrance and expressivity depending on the maternal Gal4 driver used (Fig. 2B)" - Fig. 2B doesn't seem to be a right citation for this sentence.

    Fig. 2

    • Fig. 2B: Higher magnification images of the defective cytoplasm can be shown as insets.
    • Fig. 2C: It will be helpful to indicate two other non-specific bands in the figure with arrows/arrowheads with a description in the figure legend.
    • Fig. 2E: A simple quantification of the penetrance of cuticle defects in Src42A mutants and RNAi will be helpful, as shown in Fig. S3.

    Page 9, line 9

    • This is the first time that the fast and the slow phases of germband extension are mentioned. As these two phases are used to compare the Src42A and Src42A Abl double RNAi phenotypes, they should be introduced and explained better earlier, perhaps in Introduction.

    Fig. 3

    • Fig. 3A: It will be helpful to mark the starting and the ending points of germband elongation with different markers (arrows vs. arrowheads or filled vs. empty arrowheads).
    • Fig. 3G and H should be cited in the text.
    • Fig. 3C figure legend: R2 is wrongly mentioned in Fig. 3D, E. Also, R2 (coefficient of determination) needs to be defined either in the figure legend or Materials & Methods.
    • Fig. 3D, E: statistical analysis is missing.
    • Fig. 3F: It should be mentioned that the heat map is shown for pY20 signals in the figure legend, with an intensity scale bar in the figure.

    Fig. 7A: Arrows can be added to mark the delayed germband extension.

    Fig. 8A: It should be mentioned that the heat map is shown for E-Cad signals in the figure legend, with an intensity scale bar in the figure.

    Fig. S3G: An arrowhead can be added to the gel image to indicate the band described in the legend.

    Fig. 9

    • Fig. 9A: Magnified views of the cytoplasmic clearing can be added as insets.
    • Fig. 9B: Arrow/arrowheads can be added to show the absence of the signals in the nurse cells.
    • Fig. 9C: Indicate the ending point of the germband extension by arrows.

    Page 14, lines 9-10: More explicit description of the phenotype rather than just "stronger compared to Src42Ai" will be helpful.

    Significance

    This work revealed the role of Src42A in regulating germband extension. A previous study suggested the roles of Src42A and Src64 in this developmental process using a partial loss of both proteins (Tamada et al., 2021). Using different approaches, the authors demonstrated a role of Src42A in regulating E-Cad dynamic at cell junctions during Drosophila axis elongation. Most of the analyses were done with maternal knockdown using RNAi, but they successfully generated germline clones for the first time and confirmed the RNAi phenotypes. Overall, this work contains important and exciting novel findings.

    This work will be of general interest to cell and developmental biologists, particularly researchers studying epithelial morphogenesis and junctional dynamics.

    I have expertise in Drosophila genetics, epithelial morphogenesis, imaging, and quantitative image analysis.

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    Referee #1

    Evidence, reproducibility and clarity

    Highest priority:

    1. The Src42A knockdown and germline clone experiments both cause defects in cellularization (Fig. 2B and 9A), which could result in differences in the state of the blastoderm epithelium (cell size, cell number, structural integrity, organization, etc.) between the experimental and control conditions. In addition, Src42A knockdown appears to affect the size and shape of the egg (Fig. 9A and 9C). The manuscript would be strengthened if the authors included data to demonstrate that the initial structure of the epithelium is mostly normal (quantifications of cell size, number, etc.) in the Src42A RNAi condition, as this would bolster the argument that germband extension, rather than due to indirect effects resulting from the cellularization defects. The authors may have relevant data to do this on-hand, for example using data associated with figures 1, 3, 6, and 9.
    2. There is a discrepancy in the staging of embryos used between some of the analyses, which make it hard to interpret some of the data. For example, characterization of the knockdowns in Fig. 1A and B are based on stages 10 and 15, whereas the majority of the paper is focused on earlier stages 6 - 8 during germband extension (e.g., Fig. 1D). The analysis for Fig. 1B would be more meaningful if it was done on the same stages used for subsequent phenotypic analysis so they can be directly compared.
    3. There is incongruence between figures in terms of which junctional pools (bAJs vs. tAJs) of beta-catenin and E-cadherin are quantified that makes it difficult to draw comparisons between analyses. For example pTyr levels are examined for both bAJs and tAJs in Figure 3, however, only tAJs are considered in Fig. 8. Similarly, in some cases planar cell polarity is considered (e.g., comparison of levels at AP vs DV bAJs in Fig. 6 and 9), and in other cases (e.g. Fig. 8) it is not.

    Lower priority:

    1. Introduction, 2nd paragraph - The modes of cell behaviours described to drive cell intercalation leaves out another clear example in the literature - Sun et al., 2017 - which describes a basolateral cell protrusion-based mechanism. While the authors cite this paper later, leaving it out when summarizing the state of the field misrepresents the current knowledge of the range of mechanisms responsible.
    2. 'defective cytoplasm' - this term is confusing, and could perhaps be replaced with 'cellularization defect', or something similar.
    3. Tests of statistical significance are not uniformly applied across the figures. For instance, Figures 3G + H indicate statistical significance, but Fig. 3D + E do not. Performing statistical tests throughout the paper, or clearly articulating a rationale when they are not used, would strengthen the manuscript. Specifically, the authors should consider this for Fig. 3D + E, and Fig. 7D + E, to support their arguments that rates of germband extension are different between conditions.
    4. Page 12 - "We found that Src42A showed a distinct localization at the tAJs (Fig. 1B)": Figure 1B shows a quantification of levels at bAJs, not tAJs.
    5. Figure 8 - in my opinion, using a FRAP or photoconversion approach would be a more convincing demonstration of differences in E-cadherin residency times / turnover rate than time-lapse imaging of E-cadherin:GFP alone. Authors should decide whether this improvement is worth the investment.
    6. Figure 8E - showing images of multiple tAJs, rather than z-slices of a single vertex, would better support the claim here, as the assertion is that Src42a levels are different between control and sdk RNAi conditions, and not that it varies in the z-dimension.

    Significance

    The manuscript by Backer et al. examines the function of Src42A in germband extension during Drosophila gastrulation. Prior studies in the field have shown that Src family kinases play an important role in the early embryo, including cellularization (Thomas and Wieschaus 2004), anterior midgut differentiation (Desprat et al. 2008), and germband extension (Sun et al. 2017; Tamada et al. 2021). In this study, the authors showed that Src42A was enriched at adherens junctions and was moderately enriched along junctions with myosin-II. They then showed that maternal Src42A depletion exhibits phenotypes, starting with cellularization and including a defect in germband extension. The authors focus on defects in germband extension and found that Src42A was required for timely rearrangement of junctions and that the Src42A RNAi phenotype is enhanced by Abl RNAi. Finally the authors show that E-cadherin turnover is affect by Src42A depletion.

    Overall, this study provided a higher resolution description of how Src42A regulates the behavior of junctions during germband extension. I thought the authors conclusions were well supported by the data and represent new insight in the field.

  6. Version published to 10.1101/2022.04.20.488907v1 on bioRxiv