Dynamic MicroRNA Expression Profiles During Embryonic Development Provide Novel Insights Into Cardiac Sinus Venosus/Inflow Tract Differentiation

MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic development, including the cardiovascular system. In this study, we analyze the expression profiles of different microRNAs during early cardiac development. By using whole mount in situ hybridization in developing chick embryos, with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a, miR-106a, and miR-100 expression during early stages of embryogenesis (HH3 to HH17). We also correlated those findings with putative microRNA target genes by means of mirWalk and TargetScan analyses. Our results demonstrate a dynamic expression pattern in cardiac precursor cells from the primitive streak to the cardiac looping stages for miR-23b, miR-130a, and miR-106a. Additionally, miR-100 is later detectable during cardiac looping stages (HH15-17). Interestingly, the sinus venosus/inflow tract was shown to be the most representative cardiac area for the convergent expression of the four microRNAs. Through in silico analysis we revealed that distinct Hox family members are predicted to be targeted by the above microRNAs. We also identified expression of several Hox genes in the sinus venosus at stages HH11 and HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and sinus venosus explants, we demonstrated the modulation of the different Hox clusters, Hoxa, Hoxb, Hoxc, and Hoxd genes, by these microRNAs. Furthermore, we correlated the negative modulation of several Hox genes, such as Hoxa3, Hoxa4, Hoxa5, Hoxc6, or Hoxd4. Finally, we demonstrated through a dual luciferase assay that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a, supporting a possible role of these microRNAs in Hox gene modulation during differentiation and compartmentalization of the posterior structures of the developing venous pole of the heart.

Proprotein Convertase Furina Is Required for Heart Development in Zebrafish

Cardiac looping and trabeculation are key processes during cardiac chamber maturation. However, the underlying mechanisms remain incompletely understood. Here, we report the isolation, cloning, and characterization of the proprotein convertase furina from the cardiovascular mutant loft in zebrafish. loft is an ethylnitrosourea-induced mutant and has evident defects in the cardiac outflow tract, heart looping and trabeculation, the craniofacial region, and pharyngeal arch arteries. Positional cloning revealed that furina mRNA was barely detectable in loft mutants, and loft failed to complement the TALEN-induced furina mutant pku338, confirming that furina is responsible for the loft mutant phenotypes. Mechanistic studies demonstrated that Notch reporter Tg(tp1:mCherry) signals were largely eliminated in mutant hearts, while over-expression of NICD partially rescued the mutant phenotypes, probably due to the lack of Furina-mediated cleavage processing of Notch1b proteins, the only Notch receptor expressed in the heart. Together, our data suggest a potential post-translational modification of Notch1b proteins via the proprotein convertase Furina in the heart and unveil the function of the Furina-Notch1b axis in cardiac looping and trabeculation in zebrafish and possibly in other organisms.

Twisting of the zebrafish heart tube during cardiac looping is a tbx5-dependent and tissue-intrinsic process

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.

Twisting of the heart tube during cardiac looping is a tbx5-dependent and tissue-intrinsic process

Organ laterality refers to the Left-Right (LR) asymmetry in disposition and conformation of internal organs, established in the developing embryo. The heart is the first organ to display visible LR asymmetries as it is positioned to the left side of the midline and undergoes rightward looping morphogenesis. Cardiac looping morphogenesis is tightly controlled by a combination of heart-intrinsic and -extrinsic mechanisms. As the mechanisms that drive cardiac looping are not well understood, we performed a forward genetic screen for zebrafish mutants with defective heart looping. We describe a new loss-of-function allele for tbx5a, which displays normal leftward positioning but defective rightward looping morphogenesis. By using live two-photon confocal imaging to map cardiomyocyte behavior during cardiac looping at a single-cell level we establish that during looping, ventricular and atrial cardiomyocytes rearrange in opposite directions towards the outer curvatures of the chambers. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Manipulations of cardiac looping by chemical treatment and ex vivo culture establishes that the twisting of the heart tube depends on intrinsic mechanisms and is independent from tissue growth by cell addition. Furthermore, the cardiac looping defect in tbx5a mutants is rescued in tbx5a/tbx2b double mutants, indicating that it requires proper tissue patterning. Together, our results establish that cardiac looping in zebrafish involves twisting of the chambers around the AV canal, which requires correct tissue patterning by Tbx5a.

Asymmetric Hapln1a drives regionalised cardiac ECM expansion and promotes heart morphogenesis during zebrafish development

The mature vertebrate heart develops from a simple linear cardiac tube during early development through a series of highly asymmetric morphogenetic processes including cardiac looping and chamber ballooning. While the directionality of heart morphogenesis is partly controlled by embryonic laterality signals, previous studies have suggested that these extrinsic laterality cues interact with tissue-intrinsic signals in the heart to ensure robust asymmetric cardiac morphogenesis. Using live in vivo imaging of zebrafish embryos we describe a left-sided, chamber-specific expansion of the extracellular matrix (ECM) between the myocardium and endocardium at early stages of heart morphogenesis. We use Tomo-seq, a spatial transcriptomic approach, to identify transient and regionalised expression of hyaluronan and proteoglycan link protein 1a (hapln1a), encoding an ECM cross-linking protein, in the heart tube prior to cardiac looping overlapping with regionalised ECM expansion. Loss- and gain-of-function experiments demonstrate that regionalised Hapln1a promotes heart morphogenesis through regional modulation of ECM thickness in the heart tube. Finally, we show that while induction of asymmetric hapln1a expression is independent of embryonic left-right asymmetry, these laterality cues are required to orient the hapln1a-expressing cells asymmetrically along the left-right axis of the heart tube.Together, we propose a model whereby laterality cues position hapln1a expression on the left of the heart tube, and this asymmetric Hapln1a deposition drives ECM asymmetry and subsequently promotes robust asymmetric cardiac morphogenesis.

Intrinsic cellular chirality regulates left–right symmetry breaking during cardiac looping

The vertebrate body plan is overall symmetrical but left–right (LR) asymmetric in the shape and positioning of internal organs. Although several theories have been proposed, the biophysical mechanisms underlying LR asymmetry are still unclear, especially the role of cell chirality, the LR asymmetry at the cellular level, on organ asymmetry. Here with developing chicken embryos, we examine whether intrinsic cell chirality or handedness regulates cardiac C looping. Using a recently established biomaterial-based 3D culture platform, we demonstrate that chick cardiac cells before and during C looping are intrinsically chiral and exhibit dominant clockwise rotation in vitro. We further show that cells in the developing myocardium are chiral as evident by a rightward bias of cell alignment and a rightward polarization of the Golgi complex, correlating with the direction of cardiac tube rotation. In addition, there is an LR polarized distribution of N-cadherin and myosin II in the myocardium before the onset of cardiac looping. More interestingly, the reversal of cell chirality via activation of the protein kinase C signaling pathway reverses the directionality of cardiac looping, accompanied by a reversal in cellular biases on the cardiac tube. Our results suggest that myocardial cell chirality regulates cellular LR symmetry breaking in the heart tube and the resultant directionality of cardiac looping. Our study provides evidence of an intrinsic cellular chiral bias leading to LR symmetry breaking during directional tissue rotation in vertebrate development.