Pbx4 limits heart size and fosters arch artery formation by partitioning second heart field progenitors and restricting proliferation.

Vertebrate heart development requires the integration of temporally distinct differentiating progenitors. However, few signals are understood that restrict the size of the later-differentiating outflow tract (OFT). We show that improper specification and proliferation of second heart field (SHF) progenitors in zebrafish lazarus (lzr) mutants, which lack the transcription factor Pbx4, produces enlarged hearts owing to an increase in ventricular and smooth muscle cells. Specifically, Pbx4 initially promotes the partitioning of the SHF into anterior progenitors, which contribute to the OFT, and adjacent endothelial cell progenitors, which contribute to posterior pharyngeal arches. Subsequently, Pbx4 limits SHF progenitor (SHFP) proliferation. Single cell RNA sequencing of nkx2.5+ cells revealed previously unappreciated distinct differentiation states and progenitor subpopulations that normally reside within the SHF and arterial pole of the heart. Specifically, the transcriptional profiles of Pbx4-deficient nkx2.5+ SHFPs are less distinct and display characteristics of normally discrete proliferative progenitor and anterior, differentiated cardiomyocyte populations. Therefore, our data indicate that the generation of proper OFT size and arch arteries requires Pbx-dependent stratification of unique differentiation states to facilitate both homeotic-like transformations and limit progenitor production within the SHF.

Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors.

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.

Nr2f1a balances atrial chamber and atrioventricular canal size via BMP signaling-independent and -dependent mechanisms.

Determination of appropriate chamber size is critical for normal vertebrate heart development. Although Nr2f transcription factors promote atrial maintenance and differentiation, how they determine atrial size remains unclear. Here, we demonstrate that zebrafish Nr2f1a is expressed in differentiating atrial cardiomyocytes. Zebrafish nr2f1a mutants have smaller atria due to a specific reduction in atrial cardiomyocyte (AC) number, suggesting it has similar requirements to Nr2f2 in mammals. Furthermore, the smaller atria in nr2f1a mutants are derived from distinct mechanisms that perturb AC differentiation at the chamber poles. At the venous pole, Nr2f1a enhances the rate of AC differentiation. Nr2f1a also establishes the atrial-atrioventricular canal (AVC) border through promoting the differentiation of mature ACs. Without Nr2f1a, AVC markers are expanded into the atrium, resulting in enlarged endocardial cushions (ECs). Inhibition of Bmp signaling can restore EC development, but not AC number, suggesting that Nr2f1a concomitantly coordinates atrial and AVC size through both Bmp-dependent and independent mechanisms. These findings provide insight into conserved functions of Nr2f proteins and the etiology of atrioventricular septal defects (AVSDs) associated with NR2F2 mutations in humans.

Comparative developmental analysis of Drosophila and Tribolium reveals conserved and diverged roles of abrupt in insect wing evolution.

Morphological innovation is a fundamental process in evolution, yet its molecular basis is still elusive. Acquisition of elytra, highly modified beetle forewings, is an important innovation that has driven the successful radiation of beetles. Our RNAi screening for candidate genes has identified abrupt (ab) as a potential key player in elytron evolution. In this study, we performed a series of RNA interference (RNAi) experiments in both Tribolium and Drosophila to understand the contributions of ab to the evolution of beetle elytra. We found that (i) ab is essential for proper wing vein patterning both in Tribolium and Drosophila, (ii) ab has gained a novel function in determining the unique elytron shape in the beetle lineage, (iii) unlike Hippo and Insulin, other shape determining pathways, the shape determining function of ab is specific to the elytron and not required in the hindwing, (iv) ab has a previously undescribed role in the Notch signal-associated wing formation processes, which appears to be conserved between beetles and flies. These data suggest that ab has gained a new function during elytron evolution in beetles without compromising the conserved wing-related functions. Gaining a new function without losing evolutionarily conserved functions may be a key theme in the evolution of morphologically novel structures.

Excessive feedback of Cyp26a1 promotes cell non-autonomous loss of retinoic acid signaling.

Teratogenic levels of retinoic acid (RA) signaling can cause seemingly contradictory phenotypes indicative of both increases and decreases of RA signaling. However, the mechanisms underlying these contradictory phenotypes are not completely understood. Here, we report that using a hyperactive RA receptor to enhance RA signaling in zebrafish embryos leads to defects associated with gain and loss of RA signaling. While the gain-of-function phenotypes arise from an initial increase in RA signaling, using genetic epistasis analysis we found that the loss-of-function phenotypes result from a clearing of embryonic RA that requires a rapid and dramatic increase in cyp26a1 expression. Thus, the sensitivity of cyp26a1 expression to increased RA signaling causes an overcompensation of negative feedback and loss of embryonic RA signaling. Additionally, we used blastula transplantation experiments to test if Cyp26a1, despite its cellular localization, can limit RA exposure to neighboring cells. We find that enhanced Cyp26a1 expression limits RA signaling in the local environment, thus providing the first direct evidence that Cyp26 enzymes can have cell non-autonomous consequences on RA levels within tissues. Therefore, our results provide novel insights into the teratogenic mechanisms of RA signaling and the cellular mechanisms by which Cyp26a1 expression can shape a RA gradient.

Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes of zebrafish.

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.

Sinus venosus adaptation models prolonged cardiovascular disease and reveals insights into evolutionary transitions of the vertebrate heart.

How two-chambered hearts in basal vertebrates have evolved from single-chamber hearts found in ancestral chordates remains unclear. Here, we show that the teleost sinus venosus (SV) is a chamber-like vessel comprised of an outer layer of smooth muscle cells. We find that in adult zebrafish nr2f1a mutants, which lack atria, the SV comes to physically resemble the thicker bulbus arteriosus (BA) at the arterial pole of the heart through an adaptive, hypertensive response involving smooth muscle proliferation due to aberrant hemodynamic flow. Single cell transcriptomics show that smooth muscle and endothelial cell populations within the adapting SV also take on arterial signatures. Bulk transcriptomics of the blood sinuses flanking the tunicate heart reinforce a model of greater equivalency in ancestral chordate BA and SV precursors. Our data simultaneously reveal that secondary complications from congenital heart defects can develop in adult zebrafish similar to those in humans and that the foundation of equivalency between flanking auxiliary vessels may remain latent within basal vertebrate hearts.

Stx4 is required to regulate cardiomyocyte Ca2+ handling during vertebrate cardiac development.

Requirements for vesicle fusion within the heart remain poorly understood, despite the multitude of processes that necessitate proper intracellular trafficking within cardiomyocytes. Here, we show that Syntaxin 4 (STX4), a target-Soluble N-ethylmaleimide sensitive factor attachment receptor (t-SNARE) protein, is required for normal vertebrate cardiac conduction and vesicular transport. Two patients were identified with damaging variants in STX4. A patient with a homozygous R240W missense variant displayed biventricular dilated cardiomyopathy, ectopy, and runs of non-sustained ventricular tachycardia, sensorineural hearing loss, global developmental delay, and hypotonia, while a second patient displayed severe pleiotropic abnormalities and perinatal lethality. CRISPR/Cas9-generated stx4 mutant zebrafish exhibited defects reminiscent of these patients' clinical presentations, including linearized hearts, bradycardia, otic vesicle dysgenesis, neuronal atrophy, and touch insensitivity by 3 days post fertilization. Imaging of Vamp2+ vesicles within stx4 mutant zebrafish hearts showed reduced docking to the cardiomyocyte sarcolemma. Optical mapping of the embryonic hearts coupled with pharmacological modulation of Ca2+ handling together support that zebrafish stx4 mutants have a reduction in L-type Ca2+ channel modulation. Transgenic overexpression of zebrafish Stx4R241W, analogous to the first patient's STX4R240W variant, indicated that the variant is hypomorphic. Thus, these data show an in vivo requirement for SNAREs in regulating normal embryonic cardiac function and that variants in STX4 are associated with pleiotropic human disease, including cardiomyopathy.

Ccdc103 promotes myeloid cell proliferation and migration independent of motile cilia.

The pathology of primary ciliary dyskinesia (PCD) is predominantly attributed to impairment of motile cilia. However, PCD patients also have perplexing functional defects in myeloid cells, which lack motile cilia. Here, we show that coiled-coil domain-containing protein 103 (CCDC103), one of the genes that, when mutated, is known to cause PCD, is required for the proliferation and directed migration of myeloid cells. CCDC103 is expressed in human myeloid cells, where it colocalizes with cytoplasmic microtubules. Zebrafish ccdc103/schmalhans (smh) mutants have macrophages and neutrophils with reduced proliferation, abnormally rounded cell morphology and an inability to migrate efficiently to the site of sterile wounds, all of which are consistent with a loss of cytoplasmic microtubule stability. Furthermore, we demonstrate that direct interactions between CCDC103 and sperm associated antigen 6 (SPAG6), which also promotes microtubule stability, are abrogated by CCDC103 mutations from PCD patients, and that spag6 zebrafish mutants recapitulate the myeloid defects observed in smh mutants. In summary, we have illuminated a mechanism, independent of motile cilia, to explain functional defects in myeloid cells from PCD patients. This article has an associated First Person interview with the first author of the paper.

Rdh10a Provides a Conserved Critical Step in the Synthesis of Retinoic Acid during Zebrafish Embryogenesis.

The first step in the conversion of vitamin A into retinoic acid (RA) in embryos requires retinol dehydrogenases (RDHs). Recent studies have demonstrated that RDH10 is a critical core component of the machinery that produces RA in mouse and Xenopus embryos. If the conservation of Rdh10 function in the production of RA extends to teleost embryos has not been investigated. Here, we report that zebrafish Rdh10a deficient embryos have defects consistent with loss of RA signaling, including anteriorization of the nervous system and enlarged hearts with increased cardiomyocyte number. While knockdown of Rdh10a alone produces relatively mild RA deficient phenotypes, Rdh10a can sensitize embryos to RA deficiency and enhance phenotypes observed when Aldh1a2 function is perturbed. Moreover, excess Rdh10a enhances embryonic sensitivity to retinol, which has relatively mild teratogenic effects compared to retinal and RA treatment. Performing Rdh10a regulatory expression analysis, we also demonstrate that a conserved teleost rdh10a enhancer requires Pax2 sites to drive expression in the eyes of transgenic embryos. Altogether, our results demonstrate that Rdh10a has a conserved requirement in the first step of RA production within vertebrate embryos.