The Lifeact-EGFP Quail: A New Avian Model For Studying Actin Dynamics In Vivo

  1. Yanina D. Alvarez
  2. Marise van der Spuy
  3. Jian Xiong Wang
  4. Ivar Noordstra
  5. Siew Zhuan Tan
  6. Murron Carroll
  7. Alpha S. Yap
  8. Olivier Serralbo
  9. Melanie D. White

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Abstract

Here we report the generation of a transgenic Lifeact–EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo . This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviours and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we reveal the dynamics of cells undergoing apical constriction and the emergence of large-scale actin structures such as supracellular cables and rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact–EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.

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    Reply to the reviewers

    We thank the reviewers for their time and effort in assessing our preprint. We have revised our manuscript and addressed their comments in our point-by-point response as follows:

    Reviewer 1

    The authors should cite the existing mCherry-transgenic quail lines reported by Huss et al. (2015) to compare their performance. The lines developed by Huss et al. carry multiple transgenes, and the transgene-derived fluorescence is detectable under a fluorescent stereomicroscope, which indicates that the expression of substantially high levels of fluorescent proteins in quail cells does not affect quail embryogenesis or growth.

    We have now cited the transgenic mCherry line reported by Huss et al, 2015 as an example of using live imaging of avian embryos to study development but we feel that a direct comparison between the line is invalid as the Tg(PGK1:H2B-chFP) line they report has a nuclear localised fluorescent protein and ours expresses an actin-binding fluorescent protein.

    We note that Huss et al generated three independent transgenic quail lines (Q1-3), but each only contained a single copy of the transgene (as shown in their Fig S2).

    Finally, we would like to highlight that the transgene-derived fluorescence of our Lifeact-EGFP quail line is also easily detectable under a stereomicroscope and we use this method to screen for positive Lifeact-EGFP embryos for experiments. As we show in Figure 1, the Lifeact-EGFP expression does not affect quail embryogenesis or growth.

    Here, the authors developed only a single line of single copy integration of a transgene using a weak promoter. This suggests that the procedure used by the authors to produce transgenic quail may be inefficient and that the transgene expression level is lower. The authors should present an objective measure of the transgene expression levels.

    To generate the transgenic line, we used transfection of primordial germ cells as described previously (Barzilai-Tutsch, Hila et al., eLife, 2022; Serralbo, O et al., eLife, 2020). We deliberately chose to use a UbC promoter to drive moderate expression of Lifeact to avoid potential artifacts relating to Lifeact overexpression (Courtemanche, N. et al., Nature Cell Biology, 2016; Flores, L. R. et al., Sci Rep, 2019; Spracklen, A. J. et al., Developmental Biology, 2014; Xu, R. and Du, S., Front Cell Dev Biol, 2021).

    This methodology not only generates a line with no defects in growth but it also allows us to perform high-resolution imaging and to computationally segment and quantify actin in live embryos. To objectively evaluate the transgene expression level, we have now measured the signal-to-noise ratio of Lifeact-EGFP expression and found it does not differ from that of the standard actin stain Phalloidin. We have included these measurements in Figure S1.

    Although the authors attempted to record philopodial dynamics, the images of philopodia are fuzzy. Sharper philopodial images have been published using the Huss et al. transgenic quail embryos (Sato et al., 2017), where mCherry fluorescence is widespread in the cytoplasm, which indicates no advantage of actin-associated fluorescence. (Sato Y, Nagatoshi K, Hamano A, Imamura Y, Huss D, Uchida S, Lansford R. Basal filopodia and vascular mechanical stress organize fibronectin into pillars bridging the mesoderm-endoderm gap. Development 2017; 144(2):281-291. doi.org/10.1242/dev.141259)

    The mCherry transgenic line reported by Huss et al, 2015 and used by Sato et al, 2017 ubiquitously expresses nuclear-localized mCherry fluorescent protein (Tg(PGK1:H2B-chFP)). It does not label the cytoplasm or the membrane and was used to follow cell nuclei in one set of experiments (Sato et al, 2017, Figure 6).

    The mCherry labelling of filopodia in Sato et al, 2017 was performed by DNA electroporation into wildtype embryos. Our Lifeact-EGFP transgenic line confers an advantage over this approach by 1) removing the need for electroporation to label filopodia and 2) labelling the endogenous actin that forms the filopodial structure. Although we have not optimised the imaging conditions to visualise the somitic filopodia described by Sato et al, nevertheless, we can see them quite clearly in the cross-section of our live imaging of the Lifeact-EGFP quail as demonstrated in the attached response to reviewers document.

    These filopodia, also referred to as filopodia-like protrusions (Sagar et al., Development, 2015), extend from the dorsal surface of the somites towards the ectoderm and can be seen in fixed embryos stained with Phalloidin in Figure S1 in the paper by Sato et al.

    The feasibility of live imaging is, of course, the advantage of Lifeact-EGFP; however, the actin fiber images using Lifeact-EGFP are unclear, partially because Lifeact binds to G-actin with a greater affinity than to F-actin. The authors should compare phalloidin-staining and Lifeac-EGFP on the same high-power fields of fixed specimens. The current manuscript compares staining with Lifeact-EGFP and Phalloidin-568 only under low-power magnification (Figure 1).

    We thank the reviewer for the suggestion. Although the data presented in Figure 1 are tiled images of Phalloidin-568 and Lifeact-EGFP taken on the same fixed specimens on a confocal microscope, we now also include a higher magnification image. This data clearly demonstrates the extensive overlap between Phalloidin, Lifeact-EGFP and SPY650 FastAct dye labelling (Figure 1E).

    Furthermore, we found no significant difference in signal-to-noise ratio of Lifeact-EGFP fluorescence compared to Phalloidin-568 staining (Figure S1).

    Data concerning the apical constriction indicated the versatility and limitations of the Lifeact-EGFP transgenic quail line. The transgenic mouse line carrying ZO1-EGFP transgene, better suited for analyzing the apical constriction issue and employed by Francou et al. (2023), provided cleaner data.

    The dynamics of actin during apical constriction have mostly been studied in invertebrate models where it was revealed that pulsed contractions of a medioapical actomyosin network form a ratchet-like mechanism to drive shrinkage of the apical cell area (Martin, A. C. et al., Nature, 2009; Solon, J. et al., Cell, 2009). More recently, a similar process of pulsatile apical constriction has been demonstrated in Xenopus (Christodoulou, N. and Skourides, P. A., Cell Rep, 2015) and mouse embryos (Francou, A. et al., eLife, 2023). However, the ZO1-EGFP transgenic mouse line labels tight junctions, so the dynamics of actin were inferred from staining of fixed samples by Francou et al. The Lifeact-EGFP transgenic quail line enabled us to both segment the cells and directly measure the intensity and localisation of actin as cells underwent apical constriction in a higher vertebrate embryo, providing direct information about the actin dynamics driving apical area change.

    The significance of the FRAP analysis presented in Figure 4 (F to I) is questionable. (1) The FRAP of Lifeact-EGFP that jumps between G-actin and F-actin was measured. Therefore, the data are a composite of G-actin-bound, F-actin-bound, and free transitory Lifeact-EGFP; the data do not directly reflect actin dynamics. (2) The authors should have measured FRAP at different positions in cells using smaller ROIs at the cell junction, next to the cell junction, and remote from the cell junction. (3) Because the FRAP of their measurements involves different molecular states, the recovery curve should be decomposed into individual components before discussing the difference in the recovery rates. (4) The wide range fluctuation of fluorescence intensity during the recovery process, even using a wide (4 µm × 4 µm) ROI, suggests that the fluorescence level before photobleaching was very low, which indicates a limitation in the use of the transgenic quail line with a single copy of Lifeact-EGFP.

    We apologise if the text was not clear. We did not intend to measure actin dynamics directly, but rather to compare the stability of actin at the vertices of multicellular rosettes of different orders. We used a relatively large ROI (to encompass the vertex) and measured fluorescence recovery at the vertices of lower-order (5-cell) rosettes vs higher-order (8-cell) rosettes to understand if actin stability at the vertex changes as the rosette increases in order. The fluorescence intensity level of the Lifeact-EGFP is high at the vertices of the rosettes (see Fig 4F) and the fluctuation range of fluorescence intensity during recovery was in line with what we have observed previously performing FRAP measurements in living mouse embryos (Samarage*, C.R., White*, M.D., Alvarez*, Y.A et al., Developmental Cell, 2015; Zenker*, J., White*, M. D. et al., Cell, 2018).

    To our knowledge, these are the first FRAP measurements of actin at rosette vertices.

    We have updated the text to clarify as follows:

    "To examine the stability of the actin remaining at the centre of the multicellular rosettes following contraction of the supracellular cables we used Fluorescence Recovery After Photobleaching (FRAP)."

    The authors used three wavelengths to detect fluorescence: DAPI (blue), EGFP (green), and Phaloidin-568 (red). Oddly, the authors presented the EGFP fluorescence in orange and Phaloidin-568 in gray in the pseudocolors.

    We chose to pseudocolour the images to make them accessible to people with colour blindness in accordance with current conventions.

    The data presented indicates that although Lifeact-EGFP-dependent actin labeling is useful for live imaging, its efficacy is restricted by elevated levels of background fluorescence.

    We do not find the live imaging to be restricted by high levels of background noise. Our imaging reveals an average Signal-to-Noise ratio of 1.83 +/- 0.17 (mean +/- sem) in fixed samples in Figure 1. The live imaging revealed a Signal-to-Noise ratio of 1.92 +/- 0.13 for embryos imaged in Figures 2, 3 and 4 which is comparable to the signal in the fixed embryos for both Lifeact-EGFP and Phalloidin-568.

    We can live-image the Lifeact-EGFP embryos at high resolution for extended periods (for example, tiled z-stacks at 40x magnification every 6 - 20 minutes for 4 - 10 hours) with the laser power low enough to avoid phototoxicity. Our imaging data is also of sufficient quality to allow computational segmentation with a high degree of accuracy (as demonstrated in Figures 3 and 4).

    Reviewer 2

    Alvarez and colleagues have generated a transgenic quail line expressing the popular Lifeact-eGFP reporter. This is the first actin reporter line in quail, and enables visualization and characterization of cell shapes and behaviors by following actin-rich structures. The reporter is ubiquitously expressed, and of sufficient brightness to enable high resolution live imaging. To demonstrate its usability, the authors visualized cellular protrusions and actin-rich structures during neural tube closure, migration of cardiac progenitor cells, and examined pulsatile apical constriction in the developing neuroepithelium. These results serve more as a proof-of-principle for the utility of the line rather than an in-depth analysis of any particular cell biology/mechanism, but do contain some insights and avenues for further follow-up. In general this is a nice characterization of a line that I am sure people in the avian embryo field have long been waiting for, and will be in high demand in the future.

    We thank the reviewer for their positive comments and recognition of the usefulness of the Lifeact-EGFP quail as a new model system.

    I have a few minor comments/suggestions:

    1. It would be good if the authors could elaborate on the relative photostability of the line - does it bleach quickly? Show any signs of phototoxicity?

    The photostability is dependent on the imaging conditions. In general, we have not noticed significant bleaching and there are no bleach corrections performed on the movies we show. We do not see signs of phototoxicity with the imaging conditions we are using.

    To address the photostability in more depth we examined our most challenging imaging set-ups. The high spatiotemporal imaging of lamellipodia and actin flow in Figure 1 was performed by imaging a single z-plane at 60x magnification every 5 seconds for 17.25 mins. Despite acquiring over 200 images, there was only a 9.26% loss of Lifeact-EGFP intensity during this intensive imaging.

    For the imaging of apically constricting cells in Figure 3, 4 tiled z-stacks containing 62 z-planes each were taken at 63x magnification every 5.5 mins for 110 mins. We observed an 11.8% loss of Lifeact-EGFP intensity during this time.

    This photostability is comparable to the other transgenic quail lines in our lab (Serralbo, O et al., eLife, 2020) and superior to several zebrafish and genetically modified cell lines we have imaged.

    Additionally, can the animals be maintained as homozygotes?

    The Lifeact-EGFP quails can be maintained as homozygotes and we have now indicated this in the text as follows:

    "The TgT2[UbC:Lifeact-EGFP] quails are viable, phenotypically normal and fertile and can be maintained as heterozygotes or homozygotes."

    1. Did the authors check or are they planning to verify that they did indeed have a single-integration event? Or have bred a sufficient number of generations to eliminate any potential off-target integrations?

    We have bred the Lifeact-EGFP line for enough generations that we are confident we have a single integration event that produces positive transgenics at the expected Mendelian ratio.

    1. In Figure 3: Did Lifeact-eGFP intensity and apical cell area show correlated pulsatile dynamics? They are currently shown separately over the course of constriction but it may be more convincing to show correlation analysis.

    We thank the reviewer for this excellent suggestion. We have revised Figure 3 to overlay the mean Lifeact-EGFP intensity at the apical cortex relative to the cell junctions (medial/junctional Lifeact-EGFP) and apical cell area over time for each embryo. The original separate graphs are still available in the new Figure S3A. We first established that there is a highly significant inverse correlation between medial Lifeact-EGFP intensity and apical cell area in constricting cells in each embryo (Figure S3B). We next examined the correlation between the change in medial Lifeact-EGFP intensity and the change in apical cell area for each constricting cell (Figure S3C). Although there is a high degree of variability between cells, on average we find a moderate, but highly significant correlation of 0.37 +/- 0.05, pWe have now included these results in the new Figure S3 and the text as follows:

    "Measuring the ratio of Lifeact-EGFP signal at the apical cortex relative to the cell junctions revealed an average increase of 71.7%+/- 2.9 % during the first 25% of the reduction in apical cell area (Figs. 3C, S3A-B). The inverse correlation between mean Lifeact-EGFP intensity at the apical cortex and mean apical cell area is highly significant (Fig. S3B). Furthermore, the identified cells did not undergo a constant decrease in apical cell area but instead showed a more pulsatile pattern consistent with a ratchet-like mechanism (Figs. 3C, D). There was a moderate, but highly significant correlation between the rate of change in Lifeact-EGFP intensity at the apical cortex and the change in apical cell area for individual cells (Fig. S3C)."

    1. Did they check for integrins at the filopodia tips?

    We did not check for integrins at the tips of the cardiac progenitor cell filopodia, however, we do see integrins at the tips of filopodia in other cells and these data are part of an ongoing study in our lab.

    1. In Figure 4B it is too hard for the reader to verify that these are indeed actin cables - the overlay interferes with the visualization. Could just be 10 cells coincidentally aligned. Same with Figure 4 J/K

    We have made the overlay partially transparent so that the cables are more visible. The same cable structures are also highlighted without overlays in the blue boxes in Figures 4A and 4J.

    1. Figure 4C and 4L are confusing - what is the repeated number of rosette cells mean? Are these different regions cropped out? What are the rows/columns?

    The images show the computational segmentation of the regions shown in 4A and 4J. Each panel shows the number of rosettes identified of each order (containing 5, 6, 7 or 8 cells) at t = 0h (on the left) and t = 2h (on the right).

    We initially displayed all of the rosettes on a single computational segmentation but felt it was much easier to appreciate the relative number of rosettes of each order when they are presented individually. We have updated the Figure Legend to specify that 4C and 4L show computational segmentations of the images in 4A and 4J.

    1. Time stamps on supplementary movies could be made more visible/better labelled.

    We have enlarged the timestamps on the movies.

    1. Would be helpful to include movies of the processes studied in Figures 3 and 4.

    We have now included movies showing apical constriction (Supplementary Movie 5) and rosette formation (Supplementary Movie 6).

    Reviewer 3

    The manuscript is well-written. The Lifeact-EGFP transgenic quail will be a valuable new amniote model system for in vivo investigations of the actin cytoskeleton to promote cell shape changes and tissue morphogenesis. I recommend that this manuscript be accepted with minor revisions.

    We thank the reviewer for their positive comments and are pleased they view the Lifeact-EGFP quail as a valuable new model system.

    Minor suggestions

    -Please include how many transgenic males and females were obtained from the 50 injections.

    We have now included this in the text as follows:

    "One male and one female founder were identified and mated with wild-type quails to establish lines. After further breeding the lines were indistinguishable and the line from the male founder was selected for long-term maintenance."

    -The authors state, "Cardiac progenitor cell filopodia are on average 9.1μm +/- 0.5μm long and highly dynamic with an average persistence time of 389.1 s +/- 22.9 s (n = 86 filopodia, 4 embryos). Filopodia that contact the surrounding tissues are significantly longer and more persistent than those that do not make contact (11.2μm +/- 0.7μm, n = 42 and 523.6 s +/-34.5 s, n = 37, compared to 7.2μm +/- 0.4μm, n = 44 and 276.0 s +/-20.5 s, n = 44, Fig 2C - E)."

    How does this compare to other similar cells? Does this suggest attraction, repulsion, or nothing? Does the higher filopodia persistence correlate with the cell's persistence, migration velocity or direction?

    The cardiac progenitor cell filopodia are slightly longer and more persistent on average than filopodia detected in other migrating cell types in vivo. For example, neural crest cells form filopodia that are on average 5 - 6um long and persist for 121 s in chick (Genuth, M. A. et al., Developmental Biology, 2018; McLennan, R. et al., Development, 2020) or 10um in length in zebrafish (Boer, E. F. et al., PLoS Genet, 2015). Primordial germ cells in zebrafish extend filopodia which are on average 3.4um long and persist for only 33 +/- 2.5 s (Meyen, D. et al., eLife, 2015). In Xenopus retinal ganglion cells, filopodia were on average 6.7um long and persisted for just 19 s (Blake, T. C. A. et al., Journal of cell science, 2024).

    However, the modes of migration of these cell types are quite distinct with neural crest cells collectively migrating as transiently contacting mesenchymal cells whereas primordial germ cells and retinal ganglion cells migrate individually during the embryonic stages examined. The cardiac progenitor cells form a collectively migrating epithelium which maintains cell-cell contacts and migrates over the endoderm at a speed of 4,99 +/-0.09 um min-1, so it is difficult to draw conclusions about their filopodial dynamics by comparison with other cell types characterised to date.

    The reviewer raises a very interesting question about the relationship between filopodial persistence and the migration behaviour of the individual cell. As the cardiac progenitor cells are migrating as a tightly packed collective, resolving individual cell migration behaviours is very challenging when they are homogenously labelled. To accurately correlate filopodia dynamics with individual cell migration would require highly technically demanding experiments to mosaically label the cardiac progenitor cells and track them and their filopodia dynamics live. While this would undoubtedly be an interesting experiment, we feel it is beyond the scope of the current tools manuscript.

    It is well-known that filopodia are sensors for chemotactic and haptotactic signals, and they set the direction of motility for cells. The authors rightly suggest that actin containing filopodia contact ECM components, but do not support this with any experiments.

    We agree that it would be interesting to investigate the molecular components of the filopodia more thoroughly. However, as a tools paper, our primary motivation was to present the Lifeact-EGFP transgenic quail as a new resource for the scientific community and demonstrate different applications it could be useful for - including as a new model to study filopodia dynamics in vivo.

    Significance

    The manuscript is lacking any novel insights regarding actin dynamics. In general, it would be helpful if the authors discuss the significance of their observations in more detail, especially in their Conclusion, which is brief. By carrying out more creative and insightful experiments, the authors would have offered stronger evidence for the value of the Lifeact-EGFP line to other investigators.

    The primary purpose of this manuscript was to present the Lifeact-EGFP transgenic quail as a new resource for the scientific community and demonstrate different applications it could be useful for. However, we did also make some novel insights:

    • Although neural tube protrusions have been visualised in fixed embryos for many decades, the Lifeact-EGFP transgenic quail enabled us to image them live in high spatiotemporal resolution. This revealed that they are highly dynamic, reach across the open lumen to contact each other and appear to assist in pulling the neural folds together. We also found that neural tube zippering proceeded faster in embryos with more protrusions.
    • We demonstrated that cells in the avian neuroepithelium undergo pulsatile apical constriction associated with the enrichment of medioapical actin.
    • We performed, to our knowledge, the first FRAP of actin at the vertices of multicellular rosettes and found that actin stability increases with higher rosette order.
    • Finally, we confirmed that supracellular actin cable contraction and rosette formation contribute to anisotropic bending of the neural plate during neural tube formation - a prediction made previously based on fixed tissue sections (Nishimura, T. et al., Cell, 2012) but not investigated in living avian embryos. We believe that the range of novel insights we present here demonstrates the significance of the Lifeact-EGFP transgenic quail line as a new tool for investigating vertebrate cytoskeletal dynamics and morphogenesis in vivo.

    References

    An, Y., Xue, G., Shaobo, Y., Mingxi, D., Zhou, X., Yu, W., Ishibashi, T., Zhang, L. and Yan, Y. (2017). Apical constriction is driven by a pulsatile apical myosin network in delaminating Drosophila neuroblasts. Development 144, 2153-2164.

    Barzilai-Tutsch, H., Morin, V., Toulouse, G., Chernyavskiy, O., Firth, S., Marcelle, C. and Serralbo, O. (2022). Transgenic quails reveal dynamic TCF/β-catenin signaling during avian embryonic development. eLife 11, e72098.

    Blake, T. C. A., Fox, H. M., Urbancic, V., Ravishankar, R., Wolowczyk, A., Allgeyer, E. S., Mason, J., Danuser, G. and Gallop, J. L. (2024). Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions. Journal of cell science 137.

    Boer, E. F., Howell, E. D., Schilling, T. F., Jette, C. A. and Stewart, R. A. (2015). Fascin1-dependent Filopodia are required for directional migration of a subset of neural crest cells. PLoS Genet 11, e1004946.

    Christodoulou, N. and Skourides, P. A. (2015). Cell-Autonomous Ca(2+) Flashes Elicit Pulsed Contractions of an Apical Actin Network to Drive Apical Constriction during Neural Tube Closure. Cell Rep 13, 2189-202.

    Courtemanche, N., Pollard, T. D. and Chen, Q. (2016). Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nature Cell Biology 18, 676-83.

    Flores, L. R., Keeling, M. C., Zhang, X., Sliogeryte, K. and Gavara, N. (2019). Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour. Sci Rep 9, 3241.

    Francou, A., Anderson, K. V. and Hadjantonakis, A. K. (2023). A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT. eLife 12.

    Genuth, M. A., Allen, C. D. C., Mikawa, T. and Weiner, O. D. (2018). Chick cranial neural crest cells use progressive polarity refinement, not contact inhibition of locomotion, to guide their migration. Developmental Biology 444 Suppl 1, S252-S261.

    Martin, A. C., Kaschube, M. and Wieschaus, E. F. (2009). Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457, 495-9.

    McLennan, R., McKinney, M. C., Teddy, J. M., Morrison, J. A., Kasemeier-Kulesa, J. C., Ridenour, D. A., Manthe, C. A., Giniunaite, R., Robinson, M., Baker, R. E. et al. (2020). Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia. Development 147.

    Meyen, D., Tarbashevich, K., Banisch, T. U., Wittwer, C., Reichman-Fried, M., Maugis, B., Grimaldi, C., Messerschmidt, E. M. and Raz, E. (2015). Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo. eLife 4.

    Nishimura, T., Honda, H. and Takeichi, M. (2012). Planar cell polarity links axes of spatial dynamics in neural-tube closure. Cell 149, 1084-97.

    Sagar, Prols, F., Wiegreffe, C. and Scaal, M. (2015). Communication between distant epithelial cells by filopodia-like protrusions during embryonic development. Development 142, 665-71.

    Samarage*, C. R., White*, M.D., Alvarez*, Y. D., Fierro-Gonzalez, J. C., Henon, Y., Jesudason, E. C., Bissiere, S., Fouras, A. and Plachta, N. (2015). Cortical Tension Allocates the First Inner Cells of the Mammalian Embryo. Developmental Cell 34, 435-47.

    Serralbo, O., Salgado, D., Véron, N., Cooper, C., Dejardin, M., Doran, T., Gros, J. and Marcelle, C. (2020). Transgenesis and web resources in quail. eLife 9.

    Solon, J., Kaya-Copur, A., Colombelli, J. and Brunner, D. (2009). Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell 137, 1331-42.

    Spracklen, A. J., Fagan, T. N., Lovander, K. E. and Tootle, T. L. (2014). The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis. Developmental Biology 393, 209-226.

    Xu, R. and Du, S. (2021). Overexpression of Lifeact-GFP Disrupts F-Actin Organization in Cardiomyocytes and Impairs Cardiac Function. Front Cell Dev Biol 9, 746818.

    Zenker*, J., White*, M. D., Gasnier*, M., Alvarez*, Y. D., Lim, H. Y. G., Bissiere, S., Biro, M. and Plachta, N. (2018). Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation. Cell 173, 776-791 e17.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Alvarez et al. report the generation of a transgenic Lifeact-EGFP quail line to study actin organization and dynamics in living embryos. The authors use the Lifeact-EGFP line to visualize how actin filaments guide coordinate cellular movements across tissues. Their example studies of heart and neural tube morphogenesis reveal the dynamics of cells undergoing apical constriction and the emergence of large-scale actin structures, such as supracellular cables and rosettes within the neuroepithelium.

    The manuscript is well-written. The Lifeact-EGFP transgenic quail will be a valuable new amniote model system for in vivo investigations of the actin cytoskeleton to promote cell shape changes and tissue morphogenesis. I recommend that this manuscript be accepted with minor revisions.

    Minor suggestions

    • Please include how many transgenic males and females were obtained from the 50 injections.
    • The authors state, "Cardiac progenitor cell filopodia are on average 9.1μm +/- 0.5μm long and highly dynamic with an average persistence time of 389.1 s +/- 22.9 s (n = 86 filopodia, 4 embryos). Filopodia that contact the surrounding tissues are significantly longer and more persistent than those that do not make contact (11.2μm +/- 0.7μm, n = 42 and 523.6 s +/-34.5 s, n = 37, compared to 7.2μm +/- 0.4μm, n = 44 and 276.0 s +/-20.5 s, n = 44, Fig 2C - E)."

    How does this compare to other similar cells? Does this suggest attraction, repulsion, or nothing? Does the higher filopodia persistence correlate with the cell's persistence, migration velocity or direction?

    "The tissues surrounding the cardiac progenitor cells are covered in an extracellular matrix rich in fibronectin, which also extends along some of the filopodia (Fig. S2). As integrins are known to be present at filopodial tips (Lagarrigue et al., 2015, Galbraith et al., 2007), the higher persistence of filopodia in contact with surrounding tissues may indicate a force-dependent stabilisation of the filopodia (Alieva et al., 2019). This indicates these filopodia could have signalling roles as proposed previously (Francou et., 2014) and/or mechanical roles during cardiac progenitor cell migration."

    It is well-known that filopodia are sensors for chemotactic and haptotactic signals, and they set the direction of motility for cells. The authors rightly suggest that actin containing filopodia contact ECM components, but do not support this with any experiments.

    Significance

    The manuscript is lacking any novel insights regarding actin dynamics. In general, it would be helpful if the authors discuss the significance of their observations in more detail, especially in their Conclusion, which is brief. By carrying out more creative and insightful experiments, the authors would have offered stronger evidence for the value of the Lifeact-EGFP line to other investigators.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Alvarez and colleagues have generated a transgenic quail line expressing the popular Lifeact-eGFP reporter. This is the first actin reporter line in quail, and enables visualization and characterization of cell shapes and behaviors by following actin-rich structures. The reporter is ubiquitously expressed, and of sufficient brightness to enable high resolution live imaging. To demonstrate its usability, the authors visualized cellular protrusions and actin-rich structures during neural tube closure, migration of cardiac progenitor cells, and examined pulsatile apical constriction in the developing neuroepithelium. These results serve more as a proof-of-principle for the utility of the line rather than an in-depth analysis of any particular cell biology/mechanism, but do contain some insights and avenues for further follow-up. In general this is a nice characterization of a line that I am sure people in the avian embryo field have long been waiting for, and will be in high demand in the future.

    I have a few minor comments/suggestions:

    1. It would be good if the authors could elaborate on the relative photostability of the line - does it bleach quickly? Show any signs of phototoxicity? Additionally, can the animals be maintained as homozygotes?
    2. Did the authors check or are they planning to verify that they did indeed have a single-integration event? Or have bred a sufficient number of generations to eliminate any potential off-target integrations?
    3. In Figure 3: Did Lifeact-eGFP intensity and apical cell area show correlated pulsatile dynamics? They are currently shown separately over the course of constriction but it may be more convincing to show correlation analysis.
    4. Did they check for integrins at the filopodia tips?
    5. In Figure 4B it is too hard for the reader to verify that these are indeed actin cables - the overlay interferes with the visualization. Could just be 10 cells coincidentally aligned. Same with Figure 4 J/K
    6. Figure 4C and 4L are confusing - what is the repeated number of rosette cells mean? Are these different regions cropped out? What are the rows/columns?
    7. Time stamps on supplementary movies could be made more visible/better labelled.
    8. Would be helpful to include movies of the processes studied in Figures 3 and 4.

    Significance

    Transgenic quail models are still in their relative infancy compared to more traditional/well-established model organisms, yet quail has proven to offer many new insights into developmental processes, and with its flat geometry often offers up a view of tissues and cell behaviors that can be hidden in other species. A live reporter line for actin structures is thus keenly needed by the avian developmental biology field, and this new transgenic model reported here should fill that niche nicely.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this study, the authors produced a Lifeact-EGFP transgenic quail line to investigate the cellular event dynamics that involve F-actin bundles. No perfect reagents exist to specifically label F-actin in live cells at high sensitivity; currently, Lifeact peptide may be the primary option to label actins using transgenic animals. However, it has the drawback of binding to G-actin in addition to F-actin, which results in a high background of Lifeact-EGFP fluorescence in the cytoplasm.

    A transgenic quail line was produced by lipofection of circulating PGCs with Tol2 transposon-based expression vector and Tol2 transposase expression vector; a single founder male harboring a single copy of the transgene was crossed with wild-type females to generate a transgenic colony.

    To demonstrate the utility of Lifeact-EGFP quail embryos, the authors performed the following descriptive studies: (1) filopodia extrusion; (2) actin bundle dynamics during apical constriction; (3) formation of actin bundles and multicellular rosettes; (4) FRAP analysis of actin mobility at the cellular vertices; and (5) the effect of actin polymerization inhibitors on the multicellular rosettes. The data presented demonstrate the range of utility as well as the limitations of the author's transgenic system.

    Major comments

    The authors should cite the existing mCherry-transgenic quail lines reported by Huss et al. (2015) to compare their performance. The lines developed by Huss et al. carry multiple transgenes, and the transgene-derived fluorescence is detectable under a fluorescent stereomicroscope, which indicates that the expression of substantially high levels of fluorescent proteins in quail cells does not affect quail embryogenesis or growth. Here, the authors developed only a single line of single copy integration of a transgene using a weak promoter. This suggests that the procedure used by the authors to produce transgenic quail may be inefficient and that the transgene expression level is lower. The authors should present an objective measure of the transgene expression levels. (Huss D, Benazeraf B, Wallingford A, Filla M, Yang J, Fraser SE, Lansford R. A transgenic quail model that enables dynamic imaging of amniote embryogenesis. Development 2015; 142:2850-9. doi: 10.1242/dev.121392.)

    Although the authors attempted to record philopodial dynamics, the images of philopodia are fuzzy. Sharper philopodial images have been published using the Huss et al. transgenic quail embryos (Sato et al., 2017), where mCherry fluorescence is widespread in the cytoplasm, which indicates no advantage of actin-associated fluorescence. (Sato Y, Nagatoshi K, Hamano A, Imamura Y, Huss D, Uchida S, Lansford R. Basal filopodia and vascular mechanical stress organize fibronectin into pillars bridging the mesoderm-endoderm gap. Development 2017; 144(2):281-291. doi.org/10.1242/dev.141259)

    The feasibility of live imaging is, of course, the advantage of Lifeact-EGFP; however, the actin fiber images using Lifeact-EGFP are unclear, partially because Lifeact binds to G-actin with a greater affinity than to F-actin. The authors should compare phalloidin-staining and Lifeac-EGFP on the same high-power fields of fixed specimens. The current manuscript compares staining with Lifeact-EGFP and Phalloidin-568 only under low-power magnification (Figure 1).

    Data concerning the apical constriction indicated the versatility and limitations of the Lifeact-EGFP transgenic quail line. The transgenic mouse line carrying ZO1-EGFP transgene, better suited for analyzing the apical constriction issue and employed by Francou et al. (2023), provided cleaner data.

    The significance of the FRAP analysis presented in Figure 4 (F to I) is questionable. (1) The FRAP of Lifeact-EGFP that jumps between G-actin and F-actin was measured. Therefore, the data are a composite of G-actin-bound, F-actin-bound, and free transitory Lifeact-EGFP; the data do not directly reflect actin dynamics. (2) The authors should have measured FRAP at different positions in cells using smaller ROIs at the cell junction, next to the cell junction, and remote from the cell junction. (3) Because the FRAP of their measurements involves different molecular states, the recovery curve should be decomposed into individual components before discussing the difference in the recovery rates. (4) The wide range fluctuation of fluorescence intensity during the recovery process, even using a wide (4 µm × 4 µm) ROI, suggests that the fluorescence level before photobleaching was very low, which indicates a limitation in the use of the transgenic quail line with a single copy of Lifeact-EGFP.

    Minor comment

    The authors used three wavelengths to detect fluorescence: DAPI (blue), EGFP (green), and Phaloidin-568 (red). Oddly, the authors presented the EGFP fluorescence in orange and Phaloidin-568 in gray in the pseudocolors.

    Significance

    The single Lifeact-EGFP transgenic quail line developed in this study may be useful in certain contexts; however, better lines may be obtained by checking additional lines for higher levels of transgene expression.

    The data presented indicates that although Lifeact-EGFP-dependent actin labeling is useful for live imaging, its efficacy is restricted by elevated levels of background fluorescence.

  5. Version published to 10.1101/2023.11.19.567639v3 on bioRxiv
  6. Version published to 10.1101/2023.11.19.567639v2 on bioRxiv
  7. Version published to 10.1101/2023.11.19.567639v1 on bioRxiv