Twisting of the zebrafish heart tube during cardiac looping is a tbx5-dependent and tissue-intrinsic process
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Abstract
Organ laterality refers to the left-right asymmetry in disposition and conformation of internal organs and is established during embryogenesis. The heart is the first organ to display visible left-right asymmetries through its left-sided positioning and rightward looping. Here, we present a new zebrafish loss-of-function allele for tbx5a , which displays defective rightward cardiac looping morphogenesis. By mapping individual cardiomyocyte behavior during cardiac looping, we establish that ventricular and atrial cardiomyocytes rearrange in distinct directions. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Pharmacological treatment and ex vivo culture establishes that the cardiac twisting depends on intrinsic mechanisms and is independent from cardiac growth. Furthermore, genetic experiments indicate that looping requires proper tissue patterning. We conclude that cardiac looping involves twisting of the chambers around the atrioventricular canal, which requires correct tissue patterning by Tbx5a.
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###Reviewer #3:
In this manuscript, Dr. Jeroen Bakkers and colleagues build upon their previously described cardiac-intrinsic looping of the heart, a process that is independent of the initial leftward jog of the heart that is driven by left-sided Nodal activity.
A novel allele of tbx5a is recovered in a genetic screen for mutants affecting cardiac looping subsequent to cardiac jogging. These mutants have normal gut looping, and therefore establish LR asymmetry normally. The oudegracht (oug) allele of tbx5a is molecularly more severe than the well-known heartstrings allele, and unlike hst mutants in oug mutant hearts AV canal specification is expanded. Analysis of cardiomyocyte movement within the heart between 28 to 42 hpf demonstrates a process where while ventricular CMs are displaced in a net clockwise direction (relative to the …
###Reviewer #3:
In this manuscript, Dr. Jeroen Bakkers and colleagues build upon their previously described cardiac-intrinsic looping of the heart, a process that is independent of the initial leftward jog of the heart that is driven by left-sided Nodal activity.
A novel allele of tbx5a is recovered in a genetic screen for mutants affecting cardiac looping subsequent to cardiac jogging. These mutants have normal gut looping, and therefore establish LR asymmetry normally. The oudegracht (oug) allele of tbx5a is molecularly more severe than the well-known heartstrings allele, and unlike hst mutants in oug mutant hearts AV canal specification is expanded. Analysis of cardiomyocyte movement within the heart between 28 to 42 hpf demonstrates a process where while ventricular CMs are displaced in a net clockwise direction (relative to the OFT), atrial CMs do so in a counterclockwise fashion, with distinct differences between behaviour of dorsal and ventral cells in each chamber. This movement is also evident when using transgenic lines to demarcate the early left- and right-sided myocardium of the cardiac cone, which form dorsal and ventral portions of the linear heart tube. Here the dorsal myocardium is found at the outer curvature of both chambers following looping, supporting a torsional model. In the oug mutant these differences in displacement between the dorsal and ventral aspects of the chamber are not evident, perhaps explaining looping defects that are observed. Remarkably, the authors show that the looping process can be recapitulated in explanted 24 hpf hearts, with looping not requiring further addition of second heart field-derived cells. Looping defects in oug mutants can be rescued to some extent by further loss of tbx2b, supporting a model where Tbx5 and Tbx2b act to establish chamber and AVC boundaries to promote torsional rotation of the heart and cardiac looping.
Overall this work is of a very high quality, with conclusions well supported by the evidence presented. The observations of explanted 24 hpf hearts, and demonstration of a "organ-extrinsic" process that drives looping, are of particular interest, and build well upon previously published observations.
Substantive Concerns:
Given the discrepancies observed between oug and hst mutants with respect to AVC development, have the appropriate in situs (has2, bmp4, tbx2b) been repeated in the hst background? This would be especially critical for tbx2b, given the genetic rescue experiments.
The use of hearts where heartbeat has been suppressed from 28 to 42 hpf may well affect expression of nppa and formation of the outer versus inner curvature. This should be assessed. It may well be that heartbeat and flow is affected in oug mutants as well, and that defects observed are not due only to effects on CM movement/rotation. This should be commented on, at the very least.
The analysis of cell shape (lines 320-332 and Figure 7) is highly confusing as presented. It was previously shown that left-derived CMs do not reach the OC (Figure 4K). Also, given the known requirements for cardiac contractility and shear stress to promote the elongation of OC CMs, these results are even further difficult to interpret. What is meant by "meandering" in this Figure is also not evident.
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###Reviewer #2:
Tessadori et al. address the mechanism of cardiac looping, a morphogenetic event that is essential for the generation of the different chambers of the vertebrate heart. While looping is essential for cardiac function, the complex morphogenetic events that govern this important process remain poorly understood. During the development of the two-chambered zebrafish heart, looping has been proposed to involve planar bending/buckling of the flat heart tube or torsional events that would be more similar to those involved in the formation of the helical structure of the mouse heart. In the present work, the authors use a number of elegant approaches to provide a 3-dimensional description of this process. While a recent study suggested that rotational events may be occurring at the level of the cardiac outflow tract (Lombardo …
###Reviewer #2:
Tessadori et al. address the mechanism of cardiac looping, a morphogenetic event that is essential for the generation of the different chambers of the vertebrate heart. While looping is essential for cardiac function, the complex morphogenetic events that govern this important process remain poorly understood. During the development of the two-chambered zebrafish heart, looping has been proposed to involve planar bending/buckling of the flat heart tube or torsional events that would be more similar to those involved in the formation of the helical structure of the mouse heart. In the present work, the authors use a number of elegant approaches to provide a 3-dimensional description of this process. While a recent study suggested that rotational events may be occurring at the level of the cardiac outflow tract (Lombardo et al, 2019), the present work substantially extends these findings and establishes that planar bending/buckling is only of minor importance for cardiac looping which instead depends on opposing rotational movements of the atrial and ventricular compartments that twist the heart tube around the central hinge region of the atrioventricular canal. The authors furthermore provide evidence that these morphogenetic events depend on tissue-intrinsic processes that require the function of the transcription factor tbx5a. Altogether, the present work provides important new insights into the morphogenetic events that contribute to the shaping of the zebrafish heart.
The presented experimental work is generally of very good quality and convincing evidence is presented for the different findings. While I outline below several issues that should be clarified, the authors should already have a lot of the requested information that just needs to be included. While some additional data are requested, the required experiments should all be straightforward and allow rapid improvements that would further strengthen the work.
Individual points:
In their characterization of tbx5a/oug mutants, the authors state that cardiac looping is « defective », but a precise description of the actual type of defect is lacking. From the picture in Fig.1C it looks as if looping occurs still in the right direction, but with reduced amplitude. Is this the only type of defect observed, or are there others (e.g. absent or inverted looping)? How does this phenotype compare to the previously characterized tbx5a/hst mutant (see point 2)? The authors mention/show that cardiac looping and visceral laterality are unaffected, but numbers should be included to substantiate these claims.
The authors analyse different markers of cardiac regionalization (Fig.2H) and suggest that the phenotype of tbx5a/oug mutants is different from the one previously described for tbx5a/hst (Garrity et al 2002, Camarata et al, 2010). As only oug mutant data are presented, it is however not clear to what extent the perceived differences may just be due to differences in the use / interpretation of different markers. For example Tessadori et al. talk about « Increased expression for the AV endocardial markers », which appears similar to Camarata et al. talking about « loss of AV boundary restriction » of AV marker genes. As the authors already detain the tbx5a/hst allele (used in Fig.1G) they should simply show side-by-side comparisons of marker expressions for the two mutant alleles. While the similarity or difference between oug and hst mutant phenotypes is not of major importance for the main conclusions of the paper, this point should be clarified to facilitate follow-up studies that may use either mutant to further characterize the events reported here.
In Fig. 2K & 4J the authors provide a visual representation of Z cell displacement during cardiac looping. While this is very nice, the study could be strengthened further if these data could be analysed in a more quantitative way (e.g. mean displacement index at the atrial/ventricular inner/outer curvature). This would allow us to see whether the changes observed in oug mutants are significant.
The authors report a novel spaw:GFP transgenic line that they use to label the left cardiac field. While the expression of this transgene in the left lateral plate mesoderm is expected, it is more surprising to see spaw as a marker of the left cardiac disc, as previous studies (e.g. Fig.1D of de Campos-Baptista et al, 2008) have shown spaw to be expressed to the left of the cardiac primordium, rather than within the cmlc2-positive cardiac disc itself. As the authors themselves mention in the discussion when comparing their results to Baker et al 2008 (which used myl7:GFP), it is essential to establish which cells are actually labelled by a transgene. A dorsal view of the 23 somite stage cardiac disc (e.g. spaw:GFP/myl7-RFP or GFP/cmlc2 two colour in situ) should be provided to clarify this issue.
As for spaw:GFP, the authors should provide a dorsal view of the 23 som cardiac disc to document that lft2:Gal4 is indeed specifically expressed in the left heart primordium. They should moreover clarify the orientation of the pannels in Fig.S4. E.g. Fig.S4A presents two transversal sections of the 28 hpf heart tube in which left-originating lft2-expressing cells should be located dorsally. However lft2 cells are found in the upper half of the tube in the upper section, but in the lower half in the lower section. Does this mean that the D/V orientation is inverted between the two pictures? Please clarify.
In Fig.4K and Fig.8D spaw:GFP is used to visualize left-originating cells in oug mutants. In both figures, spaw-GFP cells are located in the ventral part of transversally sectioned ventricles. I do not understand how this occurs: In wild-type animals left-originating cells initially give rise to the dorsal part of the ventricle. Through clockwise rotation of the outflow tract, these dorsal cells are then relocated to the outer curvature of the ventricle, as shown in Fig.3B. So if no rotation occurs in tbx5a/oug, why are spaw:GFP cells found in the ventral ventricle, rather than remaining in their initial dorsal position?
Sample numbers should be provided for the experiments in Fig.5C and Fig.6C.
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###Reviewer #1:
This is an original paper by Tessadori et al, showing chamber movements during zebrafish heart looping. The combination of cell tracking and genetic tracing of left markers, including with a new 0.2Intr1spaw transgene, suggests differential movements in the ventricle and atrium. Using a new mutant line for tbx5a (oug), the authors show that defective heart looping is associated with defective chamber movements. This can be rescued by inactivation of tbx2b, indicating the importance of tube patterning into chamber/avc regions. Using explant experiments and pharmacological treatments, to interfere with the tube attachment and progenitor cell ingression, the authors conclude on intrinsic mechanisms of zebrafish heart looping, with a minor contribution from planar buckling.
This study follows previous work of the team, …
###Reviewer #1:
This is an original paper by Tessadori et al, showing chamber movements during zebrafish heart looping. The combination of cell tracking and genetic tracing of left markers, including with a new 0.2Intr1spaw transgene, suggests differential movements in the ventricle and atrium. Using a new mutant line for tbx5a (oug), the authors show that defective heart looping is associated with defective chamber movements. This can be rescued by inactivation of tbx2b, indicating the importance of tube patterning into chamber/avc regions. Using explant experiments and pharmacological treatments, to interfere with the tube attachment and progenitor cell ingression, the authors conclude on intrinsic mechanisms of zebrafish heart looping, with a minor contribution from planar buckling.
This study follows previous work of the team, showing that zebrafish heart looping is independent of Nodal signaling and suggestion of intrinsic mechanisms from explant experiments. Whereas asymmetric morphogenesis has been mainly analysed in terms of direction and downstream of Nodal signaling, this work addresses the contribution of other factors to the shape of the heart loop, including chamber movements and tbx genes. It has the potential to provide a significant advance into looping mechanisms, providing that data analysis is strengthened.
Major comments
The chamber movements are interesting new observations. Yet, their analysis is currently insufficient. Although images and cell tracking have been performed in 3D, it is unclear why the quantification is flattened in 2D. In Fig. 2-4, angles are treated as linear values, whereas they should be treated as circular values using dedicated packages . In the context of the low penetrance (Fig. 1G) and variability (Fig. S2, S6) of the phenotype, the number of samples should be increased. In Fig. 2, it seems that the movement in the ventricle is towards the posterior (or venous pole), rather than the left, and so why are the movements qualified as opposite, rather than perpendicular? In addition, vectors in the dorsal/left ventricle are not opposite, so the rationale of a rotation of the ventricle is unclear. To support the claim that authors "map cardiomyocyte behavior during cardiac looping at a single-cell level", the movement of the overall chamber should be subtracted to the cell traces.
The staining of left transgenic markers is described as dorsal at 28hpf (text and Fig. 3A), and ventral at 48hpf (text and Fig. 3B) : please explain whether this implies a 180° rotation or just a general flip of the heart relative to the embryo. What is the pattern of lft2BAC in oug mutants? The legend of Fig. 9 reports "expansion of the space occupied by left-originating cardiomyocytes" : what is the percentage of the VV, VD, AV, AD regions labelled at different stages and in different experimental conditions? What is the degree of rotation of the pattern and does it correspond to that measured by cell tracking? Are markers of the inner/outer curvature (ex nppa) also rotating?
The rationale for ruling out extrinsic cues of heart looping is currently unclear. It is very difficult to compare the impact of experimental conditions impairing extrinsic cues (Fig. 5-6), without a quantitative analysis of cardiac looping and of the patterns of left-transgenic markers. No observation of the twist is provided after treatment with SU5402 in vivo. What happens with the other 8/20 embryos? A caveat of explant experiments, is that the tissue may shrink and the orientation of the sample is lost. What are the parameters of the explanted tubes (pole distance, size), and which references are used to assess patterns? The authors suggest a minor contribution of planar buckling. However, neither biological quantifications (pole distance, length of the tube axis) nor computer modelling are shown to support their views and expectations. The observation that the ventricle moves posteriorly could be compatible with a convergence of the poles, potentially contributing to looping. In Fig. 6A, it seems that pole distance is higher in oug mutants. The claim on planar buckling should be altered.
The importance of the avc is suggested by the rescue experiment with tbx2b inactivation. Yet the size and constriction of the avc is not quantified in the different experimental conditions. How are cell traces/displacement vectors in this region to support the proposal that the avc acts as a "fixed hinge"? Computer models would potentially be useful to understand the consequences of avc formation on the overall tube shape and chamber movement.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
While cardiac looping is essential for cardiac function, the complex morphogenetic events that govern this asymmetric process remain poorly understood. Asymmetric morphogenesis has been mainly analysed in terms of direction and downstream of left Nodal signaling. The work of Tessadori et al. now addresses the contribution of other factors to shape the heart loop. This manuscript builds upon a previous study from the same group, showing that cardiac looping is independent of the initial leftward jog of the …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
While cardiac looping is essential for cardiac function, the complex morphogenetic events that govern this asymmetric process remain poorly understood. Asymmetric morphogenesis has been mainly analysed in terms of direction and downstream of left Nodal signaling. The work of Tessadori et al. now addresses the contribution of other factors to shape the heart loop. This manuscript builds upon a previous study from the same group, showing that cardiac looping is independent of the initial leftward jog of the heart that is driven by left-sided Nodal activity. A recent study from another group (Lombardo et al, 2019) suggested that rotational events occur at the level of the cardiac outflow tract. The present work substantially extends these findings by providing more evidence of intrinsic mechanisms driving looping. The authors use a number of elegant approaches to provide a 3-dimensional description of this process. The presented experimental work is generally of high quality. The combination of cell tracking and genetic tracing of left markers, including with a new 0.2Intr1spaw transgene, suggests differential movements in the ventricle and atrium. A novel allele (oug), encoding a truncated version of the transcription factor tbx5a, is analysed, showing normal gut looping, indicative of normal left-right asymmetry establishment. This allele is molecularly more severe than the well-known heartstrings allele; unlike hst mutants, in oug mutant hearts specification of the atrio-ventricular canal is expanded. Oug mutants display defective heart looping, associated with defective chamber movements. This can be rescued to some extent by further loss of tbx2b, supporting a model where Tbx5a and Tbx2b act to establish chamber and atrio-ventricular canal boundaries to promote torsional rotation of the heart tube and shape the loop. Explant experiments and pharmacological treatments, to interfere with the tube attachment and progenitor cell ingression, do not prevent heart looping. Altogether, the present work provides important new insights into the morphogenetic events that contribute to the shaping of the zebrafish heart. However, there are important issues that should be addressed.
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