Defining mononuclear phagocyte distribution and behaviour in the zebrafish heart

Reviewer #1 (Public Review):

The authors effectively delineate the differential distribution and behaviour of MNPs within the heart, noting that these cells can be characterised by their expression levels of csf1ra and mpeg1.1. Key findings include the identification of distinct origins for larval macrophage populations and the sustained presence of csf1ra-expressing cells on the surface of the adult heart. The study examines the embryonic development of these MNPs, revealing that csf1ra+ cells begin populating the heart from embryonic day 3, while mpeg1.1+ cells colonise the heart around day 10, with a significant increase by day 17. Given that the emergence of mpeg1.1+ cells coincides with the reported timing for the onset of haematopoietic stem cell-derived haematopoiesis, the authors combined kaede-lineage tracing experiments and mutant backgrounds to conclude that the earliest tissue-resident macrophages in the heart are derived from primitive haematopoiesis.

The authors also note that the spatial distribution of MNPs varies, with csf1ra+ cells found on the atrium and ventricle surfaces, while mpeg1.1+ cells are initially located on the surface but later distributed throughout the cardiac tissue. Notably, the study demonstrates that tissue-resident macrophages proliferate rapidly following cardiac injury. The authors observe an increased number of proliferating csf1ra+ cells, especially in csf1ra mutant zebrafish, which likely correspond to primitive-derived tissue-resident macrophages that rapidly respond to injury and contribute to the reduced scarring observed in these mutants.

This manuscript makes an important contribution to the field by enhancing our understanding of the ontogeny of tissue-resident macrophages in the heart and their cellular behaviour in a vertebrate model capable of heart regeneration.

Strengths:

This work presents a landmark study on the ontogeny and cellular behaviour of macrophages in the zebrafish heart as it comprehensively examines their development and distribution in both embryonic and adult stages.

One of the key strengths of this study is its thorough cellular description using a range of available genetic tools. By employing transgenic lines to differentiate between a few MNP subtypes, the authors provide a detailed and nuanced understanding of these cells' origins, distribution, and behaviour. This approach allows for high-resolution characterisation of MNP populations, revealing significant insights into their potential role in cardiac homeostasis and regeneration.

Furthermore, the study's findings are significant as they parallel those observed in mouse models, thereby reinforcing the validity and relevance of the zebrafish as a model organism for studying macrophage function in the context of cardiac injury. This comparative aspect underscores the evolutionary conservation of these cellular processes and enhances the study's impact.

Another notable strength is the use of ex vivo imaging techniques, which enable the authors to observe and study the dynamic behaviour of MNPs in heart tissue in real-time. This live imaging capability is crucial for understanding how these cells interact with their environment, particularly in response to cardiac injury. The ability to visualise MNP proliferation and movement provides valuable insights into the mechanisms underlying tissue repair and regeneration.

Weaknesses:

While the manuscript offers significant insights into the ontogeny and behaviour of MNPs in the zebrafish heart, a few limitations described below should be considered:

One potential issue lies in the lineage tracing experiments using the photoconversion Tg(csf1ra:Gal4); Tg(UAS:kaede) line. The authors photoconverted all cardiac tissue macrophages present at 2 days post-fertilisation (dpf) and examined the hearts of these fish at 21 dpf. Although photoconverted macrophages were still observed at 21 dpf, the majority of cells present in the heart at that time were non-photoconverted (cyan) csf1ra+ cells. While this suggests that early-seeded embryonic csf1ra+ macrophages are retained during late larval stages, the contribution of macrophages derived from haematopoietic stem cells (HSCs) might be overestimated. An important concern is that the kaede-converted cells could have proliferated during the embryonic timeframe analysed, thereby diluting and extinguishing the converted kaede protein. This dilution effect could lead to an underestimation of the contribution of primitive embryonic macrophages relative to the HSC-derived cells, resulting in an inaccurate assessment of the proportion of embryonic-derived tissue-resident macrophages over time.

Moreover, the study reports no significant difference in immune cell numbers in the hearts of cmyb-/- mutants, which have normal primitive haematopoiesis but lack HSCs, at 5 dpf. Given the authors' suggestion that mpeg+ cells originate from the HSC wave, it is essential to assess the number of mpeg+ cells in these mutants at later stages. This assessment would clarify whether mpeg+ cells are indeed HSC-derived or if csf1ra+ cells later switch on mpeg expression. Without this additional data, conclusions about the origins of mpeg+ cells remain speculative.

The study's reliance on available genetic tools, while a strength, also introduces limitations. The use of only a few transgenic lines will not fully capture the complexity and diversity of MNP populations, leading to an incomplete understanding of their roles and dynamics.

Furthermore, while the use of ex vivo imaging provides dynamic insights into cell behaviour, it may not fully capture the complexity of in vivo conditions, possibly overlooking interactions and influences present in the living organism.

The manuscript would benefit from increasing the sample sizes to ensure the robustness of the findings. The use of Phalloidin staining to delineate single cells more accurately would also enhance the precision of cell counting and improve the overall data quality.

The study could also benefit from a more in-depth exploration of the functional consequences of MNP heterogeneity in the heart. While the cellular characterisations are thorough, the molecular and regulatory insights provided by the study are limited to a couple of RT-PCRs for some known genes.

Overall, the manuscript by Moyse and colleagues significantly advances our understanding of the ontogeny and behaviour of macrophages in the zebrafish heart, revealing important parallels with mammalian models. However, the points above should be carefully considered when interpreting the results presented in this study.

Reviewer #2 (Public Review):

In this manuscript, Moyse et. al. investigated the origins and potential functions of distinct populations of mononuclear phagocytes (MNPS) in the heart of developing and adult zebrafish. First, the authors demonstrate that the embryonic zebrafish heart contains macrophages early in development and that mpeg1.1 and csf1ra expressing macrophages vary across time and location and present that cardiac tissue macrophages (cTMs) in the juvenile heart are derived by primitive hematopoiesis. By combining the two transgenes, the authors demonstrate that there are 3 distinct (later determined to be 4) subpopulations of MNPs in adult hearts and that the distribution of these subtypes is distinct within the heart consistent with differing distributions of primitive and definitive macrophages in mammalian hearts. Further analysis of these populations demonstrates distinct morphologies of the subpopulations and analysis of markers conserved in mammals demonstrates distinct expression profiles as well. The authors go on to demonstrate that these subpopulations also demonstrate distinct behaviors via ex-vivo imaging. Lastly, the authors investigated the roles of these subpopulations in a model of cardiac injury in adult zebrafish and demonstrated that primitive-derived cTMs proliferate after injury consistent with mammalian models and that the proliferation of these macrophages likely results in reduced scarring in csf1ra mutants which have reduced recruitment of pro-inflammatory definitive macrophages. The data presented in this manuscript provides solid evidence that zebrafish MNPs behave consistently with those in mammals and further solidifies the use of zebrafish models as a useful tool in studying the role of these cells in cardiac repair and regeneration.

The data presented in this manuscript strongly supports the conclusions made by the authors and utilizes novel techniques. The authors appear to have achieved the goals they set out to investigate. The use of ex-vivo imaging to visualize the movement of these macrophage populations within the heart is especially compelling. The combined use of commonly used transgenic reporters for zebrafish macrophages is a very nice use of existing tools to address new questions and highlight the distinct populations of macrophages. While the overall manuscript is very strong and is likely to have a great impact on the field, there are a few weaknesses that should be addressed prior to acceptance:

(1) The reasoning for N used in many of these experiments is not addressed, nor is the question of the number of times experiments were performed. For purposes of rigor and reproducibility, these questions should be addressed in the methods.

(2) In investigating homologs of zebrafish and mammalian genes, the inclusion of additional classical markers and novel markers of subpopulations highlighted in numerous recent studies using single-cell RNA sequencing would greatly add to the impact.

(3) The description of the RT-PCR experiment is not included in the methods. Detailed methods should be provided including probe sequences. Additionally, a quantitative method of presenting this data would strengthen the conclusions presented here as well as the inclusion of additional markers as discussed previously.

Reviewer #3 (Public Review):

In this manuscript, Moyse et al. build on previously published data and investigate several subtypes of mononuclear phagocytes within the larval, juvenile, and adult zebrafish heart. Through the use of mpeg1.1 and csfr1a transgenic lines, the authors characterize the seeding of macrophages in the embryonic and larval heart and describe localization, proportions, morphology, and behavior of several subtypes of mpeg1.1 and csfr1a macrophages in the adult uninjured heart. The authors further provide an analysis of marker gene expression in the differing macrophage subtypes in the uninjured adult heart. Lastly, the authors perform analyses of how these populations respond to cardiac injury and show that csfr1a is important for the proportion and proliferation of these different subtypes of macrophages in the heart.

While the presence of cardiac resident macrophages and their importance in heart regeneration and cardiac disease have been extensively studied in the mouse, the same attention has only recently been given to macrophages in the adult zebrafish heart. This study provides insight into many parallels that exist between resident macrophages in the mouse and zebrafish heart, and while not especially novel, this concept is important for the zebrafish cardiac field. Overall, the conclusions of this study are mostly well supported by the data, but further analysis of marker gene expression in the various macrophage subtypes described would be an important and useful addition for zebrafish researchers studying macrophages in heart regeneration. For example, how are markers of cardiac resident macrophages (described in Wei et al, doi: 10.7554/eLife.84679) expressed in the different mpeg1.1 and csfr1a populations?