Significance Heart failure is a term used to describe the heart’s inability to pump sufficient oxygen-rich blood throughout the body. This condition is commonly caused by the loss of heart muscle cells termed cardiomyocytes that results from a heart attack. As one of the least regenerative organs in the human body, the heart fails to regenerate damaged cardiomyocytes, forms a noncontractile scar instead, and progressively fails. Unlike mammals, adult zebrafish robustly regenerate their hearts following injury through division of uninjured cardiomyocytes, thus providing an opportunity to dissect innate cardiac regenerative mechanisms. Here, we provide evidence that Notch signaling is required for cardiomyocyte division and heart regeneration in zebrafish and, therefore, highlight a genetic determinant of natural heart renewal.

The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary artery1, 2. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors3, 4. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5+ progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively5, 6. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a novel vasculogenic function to the cardiac transcription factor nkx2.5 during great vessel precursor development.

SUMMARYSecond heart field (SHF) progenitors perform essential functions during mammalian cardiogenesis. We recently identified a population of cardiac progenitor cells (CPCs) in zebrafish expressing latent TGFβ-binding protein 3 (ltbp3) that exhibits several defining characteristics of the anterior SHF in mammals. However, ltbp3 transcripts are conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF progenitors are specified in higher vertebrates. Instead, ltbp3 expression initiates at the arterial pole of the developing heart tube. Because the mechanisms of cardiac development are conserved evolutionarily, we hypothesized that zebrafish SHF specification also occurs in the ALPM. To test this hypothesis, we Cre/loxP lineage traced gata4 + and nkx2.5 + ALPM populations predicted to contain SHF progenitors, based on evolutionary conservation of ALPM patterning. Traced cells were identified in SHFderived distal ventricular myocardium and in three lineages in the outflow tract (OFT). We confirmed the extent of contributions made by ALPM nkx2.5 + cells using Kaede photoconversion. Taken together, these data demonstrate that, as in higher vertebrates, zebrafish SHF progenitors are specified within the ALPM and express nkx2.5. Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and essential role during zebrafish SHF development. Embryos injected with an nkx2.5 morpholino exhibited SHF phenotypes caused by compromised progenitor cell proliferation. Co-injecting low doses of nkx2.5 and ltbp3 morpholinos revealed a genetic interaction between these factors. Taken together, our data highlight two conserved features of zebrafish SHF development, reveal a novel genetic relationship between nkx2.5 and ltbp3, and underscore the utility of this model organism for deciphering SHF biology.KEY WORDS: Gata4, Heart development, Lineage tracing, Nkx2.5, Second heart field, ZebrafishZebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function

SUMMARYDiscovering the genetic and cellular mechanisms that drive cardiac morphogenesis remains a fundamental goal, as threedimensional architecture greatly impacts functional capacity. During development, accurately contoured chambers balloon from a primitive tube in a process characterized by regional changes in myocardial cell size and shape. How these localized changes are achieved remains elusive. Here, we show in zebrafish that microRNA-143 (miR-143) is required for chamber morphogenesis through direct repression of adducin3 (add3), which encodes an F-actin capping protein. Knockdown of miR-143 or disruption of the miR-143-add3 interaction inhibits ventricular cardiomyocyte F-actin remodeling, which blocks their normal growth and elongation and leads to ventricular collapse and decreased contractility. Using mosaic analyses, we find that miR-143 and add3 act cell-autonomously to control F-actin dynamics and cell morphology. As proper chamber emergence relies on precise control of cytoskeletal polymerization, Add3 represents an attractive target to be fine-tuned by both uniform signals, such as miR-143, and undiscovered localized signals. Together, our data uncover the miR-143-add3 genetic pathway as essential for cardiac chamber formation and function through active adjustment of myocardial cell morphology.

During mammalian embryogenesis, cardiac progenitor cells constituting the second heart field (SHF) give rise to the right ventricle and primitive outflow tract (OFT). In zebrafish, previous lineage-tracing and mutant analyses suggested that SHF ventricular and OFT progenitors co-migrate to the arterial pole of the zebrafish heart tube soon after their specification in the field of anterior lateral plate mesoderm (ALPM). Using additional prospective lineage tracing, we demonstrate that while SHF ventricular progenitors migrate directly to the arterial pole, OFT progenitors become temporarily sequestered in the mesodermal cores of pharyngeal arch 2 (PA2), where they downregulate expression. While there, they intermingle with precursors for PA2-derived head muscles (HMs) and hypobranchial artery endothelium, which we demonstrate are co-specified with SHF progenitors in the ALPM. Soon after their sequestration in PA2, OFT progenitors migrate to the arterial pole of the heart and differentiate into OFT lineages. Lastly, we demonstrate that SHF ventricular and OFT progenitors exhibit unique sensitivities to a mutation in Our data highlight novel aspects of SHF, OFT and HM development in zebrafish that will inform mechanistic interpretations of cardiopharyngeal phenotypes in zebrafish models of human congenital disorders.

Background The mammalian outflow tract (OFT) and primitive right ventricle arise by accretion of newly differentiated cells to the arterial pole of the heart tube from multi-potent progenitor cells of the second heart field (SHF). While mounting evidence suggests that the genetic pathways regulating SHF development are highly conserved in zebrafish, this topic remains an active area of investigation. Results Here, we extend previous observations demonstrating that zebrafish tbx1 (van gogh, vgo) mutants show conotruncal defects consistent with a conserved role in SHF-mediated cardiogenesis. Surprisingly, we reveal through double in situ analyses that tbx1 transcripts are excluded from cardiac progenitor cells or differentiated cardiomyocytes, suggesting a non-autonomous role in SHF development. Further, we find that the diminuitive ventricle in vgo animals results from a 25% decrease in cardiomyocyte numbers that occurs subseqent to heart tube stages. Lastly, we report that although SHF progenitors are specified in the absence of Tbx1, they fail to be maintained due to compromised SHF progenitor cell proliferation. Conclusion These studies highlight conservation of the Tbx1 program in zebrafish SHF biology.

Highlights d One-photon calcium imaging of brain activity can suffer from neuropil crosstalk d Targeting GCaMPs to the cell body reduces neuropil crosstalk d One-photon imaging of somatic GCaMP reduces artifactual spikes and correlations d Somatic GCaMPs can be used in multiple species, such as mice and zebrafish

SUMMARYMicrodeletions involving TBX1 result in variable congenital malformations known collectively as 22q11.2 deletion syndrome (22q11.2DS). Tbx1-deficient mice and zebrafish recapitulate several disease phenotypes, including pharyngeal arch artery (PAA), head muscle (HM), and cardiac outflow tract (OFT) deficiencies. In zebrafish, these structures arise from nkx2.5+ progenitors in pharyngeal arches 2–6. Because pharyngeal arch morphogenesis is compromised in Tbx1-deficient animals, the malformations were considered secondary. Here, we report that the PAA, HM, and OFT phenotypes in tbx1 mutant zebrafish are primary and arise prior to pharyngeal arch morphogenesis from failed specification of the nkx2.5+ pharyngeal lineage. Through in situ analysis and lineage tracing, we reveal that nkx2.5 and tbx1 are co-expressed in this progenitor population. Furthermore, we present evidence suggesting that gdf3-ALK4 signaling is a downstream mediator of nkx2.5+ pharyngeal lineage specification. Collectively, these studies support a cellular mechanism potentially underlying the cardiovascular and cranio-facial defects observed in the 22q11.2DS population.