Physical inputs orchestrate cellular dynamics to sculpt organs of various morphologies. In the embryonic heart, for example, functional cues engendered by blood flow and contractility regulate cell size, shape and number to create the curvatures of the ventricular chamber. However, we have limited understanding of the many ways by which forces associated with function integrate diverse cellular mechanisms in multiple tissues to build a composed organ. Here, we leverage the junctional location and precise dimensions of the cardiac outflow tract (OFT), a cylindrical carrier of blood between the heart and the vasculature, to address this question. Since the OFT is formed by late-differentiating second heart field cells annexing to the arterial pole of a contractile heart tube, we hypothesize that function could influence the earliest steps of OFT development. Using high-resolution morphometrics in the optically accessible zebrafish embryo, we show that in the context of normal cardiac function, the OFT expands via concerted accumulation of inner endocardial and outer myocardial cells. However, in mutants with disrupted cardiac function, OFT endocardial growth ceases and the arrangement of the overlying myocardial cells is altered. By evaluating patterns of cell behavior in both wild-type and mutant embryos, we identify essential roles for cardiac function in promoting both the proliferation of the OFT endocardium and the addition of endothelial cells to the OFT from the adjacent aortic arches. Intriguingly, loss-of-function of the TGFβ receptor Alk1 leads to reduced addition of endothelial cells to the OFT endocardium without inhibiting OFT endocardial proliferation, suggesting that these two endocardial cell behaviors can be uncoupled through molecular, and possibly physical, mechanisms. Overall, our studies illuminate the cellular basis for essential early phases of OFT morphogenesis, while also pointing toward molecular mechanisms by which cardiac function shapes OFT development.

The mature heart is comprised of multiple types of specialized cardiomyocytes, each with distinct functional attributes. However, the mechanisms that specify discrete populations of cardiac progenitors are not well understood. For example, it is clear that cardiac pacemaking activity is confined to a specialized population of cells in the cardiac venous pole, but the signals that create the appropriate number of pacemaker cardiomyocytes remain unknown. We have therefore sought to understand how pacemaker cells develop in the zebrafish embryo. First, we have investigated pacemaker cells in the inflow tract (IFT) of wild-type zebrafish embryos. We have observed that IFT cardiomyocytes express a suite of molecular markers that are reminiscent of mammalian pacemaker cells and that confer attributes specific to this population. Furthermore, we have determined that IFT progenitors are localized to discrete areas at the edges of the heart fields, prior to their differentiation. Next, we have shown that the specification of this IFT progenitor population is influenced by opposing inputs from two signaling pathways: Hedgehog (Hh) signaling and Bmp signaling. Given our prior finding that Hh signaling promotes cardiomyocyte production, we were surprised to discover that Hh signaling also acts to delimit the number of IFT cardiomyocytes. Using both genetic and pharmacological manipulations of the Hh pathway, we have shown that loss of Hh signaling results in dramatically expanded expression of IFT markers. Conversely, Bmp signaling drives IFT formation, as embryos with reduced Bmp signaling have a diminished IFT. Timed manipulations of Hh and Bmp activity have demonstrated that both signals act during early steps of cardiac patterning to define IFT size. Intriguingly, reducing both Hh and Bmp signaling restores a nearly normal number of IFT cells. We therefore propose a model in which IFT specification relies on both limited amounts of Hh signaling and robust levels of Bmp signaling, which together set appropriate boundaries for the IFT progenitor population. Our findings reveal novel mechanisms of cardiac patterning; in the long term, these studies could contribute to our understanding of congenital heart disease and improve efforts to generate pacemaker cells in vitro for use in regenerative medicine.

The vertebrate heart is comprised of two types of chambers, ventricles and atria, which exhibit unique structural and contractile properties. Effective cardiac function depends upon the distinct morphological, physiological, and molecular characteristics of ventricular and atrial cardiomyocytes. Intriguingly, despite the early specification of ventricular and atrial lineages, chamber-specific features need to be actively reinforced even after myocardial differentiation is underway. By combining genetic and embryological approaches, work in zebrafish has revealed multiple players with potent influences on chamber fate specification and commitment. Notably, our prior studies in zebrafish have shown that sustained FGF signaling acts upstream of nkx2.5 to maintain ventricular chamber identity: upon inhibition of FGF signaling or loss of nkx2.5 function, ventricular cardiomyocytes extinguish expression of the ventricular gene vmhc and initiate ectopic expression of the atrial gene amhc. However, the rest of the pathway acting downstream of FGF signaling in this context remains largely unknown. Here, we show that MEK1/2-ERK1/2 signaling plays an important part in promoting ventricular identity maintenance. Pharmacological inhibition of MEK signaling results in ectopic amhc expression as well as reduced vmhc expression in ventricular cardiomyocytes. Epistasis analysis suggests that MEK signaling influences ventricular identity maintenance downstream of FGF signaling and upstream of nkx2.5 function. We find that FGF signaling and MEK signaling both influence ventricular identity maintenance over a similar timeframe, and we can detect phosphorylated ERK (pERK) in the myocardium during these stages. Intriguingly, the role of FGF-MEK activity seems to be context-dependent: some regions of the ventricle are more sensitive than others to inhibition of the FGF-MEK pathway. On the other hand, in the atrium, although endogenous levels of MEK-ERK signaling activity are not sufficient to induce ventricular traits in atrial cardiomyocytes, heightened MEK1 activity can provoke ectopic expression of vmhc. Together, our data indicate distinct functions of MEK-ERK signaling in the maintenance of ventricular and atrial identities.

The atrial and ventricular chambers of the heart behave as distinct

functional subunits with unique morphological, contractile, and electrophysiological properties. Therefore, the proper differentiation of atrial and ventricular tissues is crucial for the formation of a functional heart. Although the differences between atrial and ventricular chambers are well characterized, less is known about how these chamber-specific attributes are acquired. Here, we show that the FGF signaling pathway plays an essential part in regulating ventricular chamber identity. Loss of FGF signaling following the onset of ventricular differentiation results in gradual accumulation of atrial cells, a corresponding loss of ventricular cells, and a progressive increase in the appearance of ectopic atrial cells within the ventricle. These phenotypes reflect an important role for sustained FGF signaling in maintaining chamber- specific characteristics in ventricular cardiomyocytes. In addition, FGF signaling is required to establish ventricular character in late-differentiating cardiomyocytes that append to the arterial pole of the heart. Our results highlight that, even after the initial specification of atrial and ventricular fates, ventricular identity needs to be continuously reinforced. Our studies suggest active maintenance as a pervasive developmental mechanism by which cell fates are enforced until a cell is committed. In addition, we present our findings from a phenotype-driven small molecule screen to find additional signaling pathways that modulate atrial and ventricular identities.

Cardiac morphogenesis and skeletal muscle morphogenesis are critical developmental processes that require ongoing coordination and signaling between cells and the matrix of their surrounding environment. The extracellular matrix (ECM) itself is a complex and dynamic structure whose regulation is of the utmost importance to cell behavior. While it is well characterized that the ECM is essential for organogenesis, it is less known how ECM composition informs development. Here, we show that the transmembrane protein Tmem2 is an essential regulator of ECM organization during cardiac and skeletal muscle morphogenesis. In zebrafish, maternal and zygotic depletion of tmem2 renders embryos unable to form primitive heart tubes, whereas in muscle tissue this same loss results in destabilization of muscle fibers. In contrast, zygotic loss of tmem2 results in ectopic atrioventricular canal (AVC) differentiation. All three of these striking phenotypes are accompanied by disorganized and defective ECM deposition, reflecting an important role for Tmem2 in regulation of the extracellular environments that impact development. The molecular mechanism of Tmem2 function, however, is unclear. Our results identify the structural requirements for Tmem2 activity across multiple developmental roles. Further, our studies characterize disorganization of the ECM in tmem2 mutants. Finally, our findings support the model that Tmem2 directly influences ECM structure and composition in a context-dependent manner, while maintaining the possibility that Tmem2 can have additional mechanisms of activity.

Development of the vertebrate heart involves the collaboration between an array of different cell types, each engaging in a meticulously coordinated symphony of spatially and temporally regulated morphogenetic behaviors. Even small errors in cardiac morphogenesis can have severe physiological consequences. In an effort to understand the regulators of cardiac morphogenesis, prior studies have examined the regionalized changes in cell morphology accompany chamber curvature formation. However, we do not yet fully understand the subcellular mechanisms that drive these cell shape changes. Prior studies have also indicated that actomyosin dynamics are important for chamber curvature morphogenesis, and multiple groups have observed differential organization of F-actin in cardiomyocytes of the inner and outer chamber curvatures. Here, we aimed to develop a comprehensive approach to identify distinctions between the actomyosin organization in inner and outer curvature cardiomyocytes that might promote regional changes in cell morphologies. We show that the actomyosin network indeed exhibits differential patterns of organization between cardiomyocytes in the inner and outer ventricular curvatures. We also find that tbx5a mutants, which fail to undergo proper curvature formation, fail to develop curvature-specific patterns of subcellular actomyosin organization. From these data, we propose a model in which the acquisition of regionalized actomyosin organization patterns drives curvature formation by promoting the development of curvature-specific cell morphologies.

Vertebrate heart formation requires input from two temporally distinct phases of cardiomyocyte differentiation. Progenitors from the first heart field form the primitive heart tube in the initial phase. Then, in a subsequent phase, progenitors from the second heart field contribute additional cells to both poles of the heart. Recruitment of second heart field progenitor cells to the arterial pole creates the cardiac outflow tract (OFT), but the precise mechanisms responsible for controlling OFT progenitor production remain to be elucidated. Members of the Cadm family of cell adhesion molecules participate in both homophilic and heterophilic binding to regulate intercellular interactions and signaling. Previously, our laboratory had demonstrated that morpholino knockdown of cadm4 results in a dramatic OFT expansion, which is preceded by an increase in OFT progenitors. In order to extend our studies of Cadm4 function and to analyze its potential interactions with other Cadm family members, we have generated mutant alleles of cadm4, cadm2a, and cadm3 and analyzed their effects on OFT formation. Evaluation of OFT morphology and the OFT progenitor population in cadm4 mutants did not reveal significant defects in OFT development, in contrast with the cadm4 morphant phenotype. Similarly, we found that OFT formation was normal in cadm2a mutants, cadm3 mutants, cadm4;cadm2a double mutants, and cadm4;cadm3 double mutants. Altogether, our findings document a discrepancy between the phenotypes of cadm4 mutants and cadm4 morphants and lay the groundwork for future studies on whether and how Cadm molecules regulate OFT formation.

The rhythmic contraction of the heart depends upon the conductive properties of a small population of highly specialized cardiomyocytes, known as pacemaker cells. In the early embryonic heart, pacemaker cells reside in a ring-like pattern at the venous pole of the atrial chamber, in a region known as the inflow tract (IFT). While the gene regulatory networks that contribute to pacemaker cell differentiation have been previously investigated, we do not fully understand the upstream signaling landscape that defines the dimensions of the IFT. Our analyses in zebrafish suggest an essential role for Bmp signaling in promoting the formation of an appropriate number of IFT cardiomyocytes. Diminished Bmp signaling activity in alk8 mutants results in a substantial reduction of the number of IFT cells. Oppositely, heightened levels of Bmp signaling in chordin mutants leads to an increased number of IFT cells. In both alk8 and chordin mutants, altered numbers of IFT cells are evident within the primitive heart tube, indicating that Bmp signaling impacts IFT development prior to heart tube formation. Indeed, treatment with DMH1, a pharmacological antagonist of Bmp signaling, during gastrulation results in a significant reduction of IFT cell number, but treatment with DMH1 during early somitogenesis or during heart tube formation does not alter the number of IFT cells. Intriguingly, pharmacological inhibition of Wnt signaling during IFT cell differentiation suppresses the enlargement of the IFT in chordin mutants, implying that Wnt signaling acts downstream of Bmp signaling during IFT development. Together, these data suggest a model in which Bmp signaling acts during IFT progenitor specification, upstream of Wnt-directed IFT differentiation, to produce an appropriate number of IFT cardiomyocytes.

Neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) is a member of the NEDD4 family of HECT E3 ubiquitin ligases. NEDD4 is highly expressed in cardiomyocytes, but its specific function in adult cardiomyocytes is unknown. To study the role of NEDD4 in adult cardiomyocytes, we have generated a Nedd4 floxed mouse model and crossed it with tamoxifen-inducible aMHC-MerCreMer mice to generate an inducible Nedd4 cardiomyocyte-specific knockout mouse model (Nedd4 icKO). In this study, we performed comprehensive molecular and cardiac physiological studies on Nedd4 icKO mice. Our results show that after performing age-dependent studies, cardiac function and structure were not dramatically altered in Nedd4 icKO mice, suggesting that the loss of NEDD4 did not cause cardiomyopathy. Further experiments revealed that the levels of chaperone proteins and the levels of global ubiquitination were not changed within the cardiac tissue of Nedd4 icKO mice. Together, these results suggest that deletion of NEDD4 in adult cardiomyocytes does not cause overt cardiomyopathy.

Cyclin-dependent kinase-like 5 (CDKL5) syndrome is a neurodevelopmental disorder that results in early-onset intractable seizures, mental retardation, and a host of autistic phenotypes. Although the role of CDKL5 is not well understood, previous studies have implicated it plays a crucial role within the AKT/mTOR pathways, which regulate cellular proliferation, apoptosis, and synaptogenesis. This study focused on generating neural progenitor cells (NPCs) and subsequently differentiating them into neurons, for the purpose of modeling the effects of CDKL5 mutations on the phenotypes listed above. hiPSC-derived CDKL5 NPCs exhibited decreased cell proliferation and increased apoptosis as compared to controls, which is congruent with the findings of previous studies with regards to inactivation of the AKT/mTOR pathways. Furthermore, hiPSC-derived CDKL5 neurons displayed no change in total dendritic length but appeared to grow more slowly during early neurodevelopment as compared to controls. These neurons also had a greater density of dendritic spines, with increased motility, as compared to controls. These findings conflict with the results from previous CDKL5 studies based on mouse knockout models. The discrepancy in results suggests that CDKL5 function may not be preserved across species, a claim that is supported by the fact that the murine models do not recapitulate the defining feature of CDKL5: early-onset intractable seizures.