Semaphorin3f as an intrinsic regulator of chamber-specific heart development
Posted on: 30 July 2021
Preprint posted on 19 May 2021
Categories: cell biology, developmental biology, genetics
Background
The heart is the first organ to form and function during embryogenesis and incomplete or altered cardiogenesis can lead to congenital heart disease (CHD). Zebrafish has emerged as a powerful model to study heart development because their embryos develop externally and transparently, which allows scientists to monitor major events during cardiac morphogenesis1.
Early zebrafish heart composes of two main cell types: endocardial cells and cardiomyocytes. After 15 hours post-fertilization, the bilateral cardiac crescent starts to migrate and fuse at the midline, which gives rise to a linear heart tube. The heart tube, then, undergoes twisting and expansion to specify the primitive atrial and ventricular chambers, which are later separated by atrioventricular valves1. During chamber specification, the cardiomyocytes acquire new identities to match their distinct physiological functions in the atria and ventricle2,3,4. How do cardiomyocytes recognize their residing location? Many extrinsic and intrinsic factors coordinate this process, of which the fibroblast growth factor (Fgf) is a well-studied example. Previous research has shown that loss of Fgf signaling causes the accumulation of an atrial marker (myh6) in the ventricular area2,3,4.
Initially involved in axon guidance, Semaphorin signaling becomes widely appreciated in other cellular contexts such as angiogenesis and organogenesis. There are four classes of Semaphorins (Sema3s – 7s) in vertebrates. Among these, only the Sema3 class produces the secreted form of ligands. Sema3 ligands bind and mediate the signals through the canonical Plexin receptors and Neuropilins (Nrp) coreceptors5. It has been shown that Sema3s serve as attractant cues for neural crest cells migrating into the developing heart. However, the function of Sema3s in heart cell differentiation is unknown. In this preprint, the authors address whether Sema3s are involved in cardiomyocyte spatial specification.
Key findings
1. Sem3fb mutants show edema and cardiac malfunction
The authors examined the expression of Sema3fb throughout heart looping and chamber morphogenesis using in situ hybridization technique (ISH). At 24 to 48 hours post-fertilization (hpf), Sema3fb selectively expresses in cardiomyocytes through the entire heart tube. ISH of Sema3fb in transgenic zebrafish Tg(Flk:EGFP) with GFP labeling endocardial cells showed that Sem3fb does not express in the endocardium. To generate sema3fb genetic mutants, authors used CRISPR/Cas9 genome editing with guide RNAs targeting exon 2 in the sema3fb gene, which produced two allelic variants sema3fbca305, and sema3fbca306. Sema3fbca305 contains 19 base pair (bp) deletion next to ATG starting site, while sema3fbca305 harbors a 10 bp insertion at the same region. Both variants produce a truncated and non-functional sema3fb.
From 48 hpf, sema3fb mutants start to show cardiac oedema. At 72 hpf, mutants exhibit cardiac malfunction with a slight reduction in ventricular volume (systole/diastole), stroke volume, cardiac output, ejection fraction, and heart rate. The authors predicted that the failure of the heart valve causes a retrograde flow, resulting in an accumulation of fluid in the pericardial cavity. Indeed, the atrioventricular valves of the sema3fb mutants are underdeveloped comparing to wild-type counterparts.
2. Sema3fb – Plxna3 ligand/receptor regulates cardiomyocyte specification at the boundary between atria and ventricle
Cardiac chamber morphogenesis is completed by 48 hpf with the establishment of a defined ventricle and atria. Comparing to the wild type, sema3fb mutants have a smaller atrial and ventricle chambers. This was not due to the defect in either apoptosis or proliferation process and was instead caused by a significant decrease in cardiomyocyte size.
The transition between the ventricular and atrial area is marked by a constriction border where the atrioventricular valve will develop. Cardiomyocytes locating at the ventricle are distinguished from the atria via the expression of myh7 and myh6, respectively. A careful characterization of sema3f mutants revealed a disproportion between two chambers. In sema3fb mutants, myh7 level decreased in the ventricular area while myh6 expression expanded over the constriction border (Figure 1). Also, bmp4, another marker of the atrioventricular canal, failed to restrict to the border. These results suggest that the boundary between the two chambers forms irregularly. Sema3fb mediates cellular signals through the plxna3 receptor and nrp2b coreceptors. Morpholino–mediated knockdown of plxna3 recapitulates sema3fb phenotype. Interestingly, overexpression of sema3fb does not resolve the bmp4 restriction pattern, which indicates that an appropriate amount of sem3fb ligand is required to fine-tunes the boundary between two chambers.
Figure 1. In situ hybridization staining of myh7(E-G) and myh6 (I-K) in wild-type and sema3fb mutants. Images were obtained from Figure 5 (E-K) in the R Halabi et al. Preprint. DOI: https://doi.org/10.1101/2021.05.19.444704.
What I like about the preprint
The preprint has beautifully dissected the effect of sema3fb – plxn3a on cardiac chamber morphogenesis. The authors used both gain-of-function and loss-of-function zebrafish models to prove that either too low or too high a level of sema3fb could interfere with the constriction border between ventricle and atria. The secreted class 3 Semaphorins (Sema3s) are highly versatile ligands. Here, the authors suggest sema3f plays a new role in cardiomyocyte specification and spatial restriction. Similar methods and approaches could be used to study other members of Semaphorin classes during cardiac development.
Questions to authors:
- Given that Semaphorin classes and their receptors are highly diverse. Semaphorin class 3 alone contains seven members. Knockout mutants of each member impose different cardiovascular defects5. I am curious about the initial reasons for the authors to focus on studying sema3fb during heart development. Could you tell us more about why?
- The preprint has shown that a proper amount of sema3fb and the spatial restriction of the plxn3a receptor are critical to set the boundary between atrial and ventricle. It will be interesting to know how the expression of sema3fb and plxn3a is regulated during heart development?
Acknowledgment
I would like to thank Dr. Osvaldo Conteras (Victor Chang Cardiac Institute, Australia) for proofreading the highlight.
Reference:
- Liu J., Stainier D.Y.R., Zebrafish in the study of early cardiac development, Circulation Research,
- Marques S.R., Lee Y., Poss K. D., Yelon D., Reiterative roles for FGF signaling in the establishment of size and proportion of the zebrafish heart, Developmental Biology, 2008.
- Pradhan A. et al., FGF signaling reinforces cardiac chamber identity in the developing ventricle, Development, 2017
- Guo Y., Pu W.T., Cardiomyocyte maturation new phase in development, Circulation Research, 2020.
- Epstein J., Aghajanian H., Singh M. K, Semaphorin signaling in cardiovascular development, Cell metabolism, 2015.
doi: https://doi.org/10.1242/prelights.30191
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