A Novel Perspective on Neuronal Control of Anatomical Patterning, Remodeling, and Maintenance.

While the nervous system may be best known as the sensory communication center of an organism, recent research has revealed a myriad of multifaceted roles for both the CNS and PNS from early development to adult regeneration and remodeling. These systems work to orchestrate tissue pattern formation during embryonic development and continue shaping pattering through transitional periods such as metamorphosis and growth. During periods of injury or wounding, the nervous system has also been shown to influence remodeling and wound healing. The neuronal mechanisms responsible for these events are largely conserved across species, suggesting this evidence may be important in understanding and resolving many human defects and diseases. By unraveling these diverse roles, this paper highlights the necessity of broadening our perspective on the nervous system beyond its conventional functions. A comprehensive understanding of the complex interactions and contributions of the nervous system throughout development and adulthood has the potential to revolutionize therapeutic strategies and open new avenues for regenerative medicine and tissue engineering. This review highlights an important role for the nervous system during the patterning and maintenance of complex tissues and provides a potential avenue for advancing biomedical applications.

Use of the Nutrition Environment Measures Survey: A Systematic Review.

INTRODUCTION: The Nutrition Environment Measurement Survey (NEMS) measures were developed to assess the consumer food environment in stores and restaurants. NEMS tools have been widely used in research and adapted for diverse settings and populations in the 15 years since they were created. This systematic review examines the use and adaptations of these measures and what can be learned from published studies using NEMS tools. METHODS: A comprehensive search of bibliographic databases was conducted from 2007 to September 2021, supplemented by backward searches and communications with authors, to identify research articles using NEMS tools. Data on purpose, key findings, sample characteristics, NEMS characteristics, and modifications were abstracted. Articles were categorized on the basis of study goals, NEMS tool(s) used, variables measured, and common themes. RESULTS: A total of 190 articles from 18 countries were identified. Most studies (69.5%, n=123) used a modified version of NEMS tools. There were 23 intervention studies that used measures from NEMS tools or adaptations as outcomes, moderators, or process assessments. A total of 41% (n=78) of the articles evaluated inter-rater reliability, and 17% (n=33) evaluated test-retest reliability. DISCUSSION: NEMS measures have played an important role in the growth of research on food environments and have helped researchers to explore the relationships among healthy food availability, demographic variables, eating behaviors, health outcomes, and intervention-driven changes in food environments. The food environment is constantly changing, so NEMS measures should continue to evolve. Researchers should document data quality of modifications and use in new settings.

PFAS in drinking water and serum of the people of a southeast Alaska community: A pilot study.

Per- and polyfluoroalkyl substances (PFAS) have become a target of rigorous scientific research due to their ubiquitous nature and adverse health effects. However, there are still gaps in knowledge about their environmental fate and health implications. More attention is needed for remote locations with source exposures. This study focuses on assessing PFAS exposure in Gustavus, a small Alaska community, located near a significant PFAS source from airport operations and fire training sites. Residential water (n = 25) and serum (n = 40) samples were collected from Gustavus residents and analyzed for 39 PFAS compounds. In addition, two water samples were collected from the previously identified PFAS source near the community. Fourteen distinct PFAS were detected in Gustavus water samples, including 6 perfluorinated carboxylic acids (PFCAs), 7 perfluorosulfonic acids (PFSAs), and 1 fluorotelomer sulfonate (FTS). ΣPFAS concentrations in residential drinking water ranged from not detected to 120 ng/L. High ΣPFAS levels were detected in two source samples collected from the Gustavus Department of Transportation (14,600 ng/L) and the Gustavus Airport (228 ng/L), confirming these two locations as a nearby major source of PFAS contamination. Seventeen PFAS were detected in serum and ΣPFAS concentrations ranged from 0.0170 to 13.1 ng/mL (median 0.0823 ng/mL). Perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) were the most abundant PFAS in both water and serum samples and comprised up to 70% of ΣPFAS concentrations in these samples. Spearman's correlation analysis revealed PFAS concentrations in water and sera were significantly and positively correlated (r = 0.495; p = 0.0192). Our results confirm a presence of a significant PFAS source near Gustavus, Alaska and suggest that contaminated drinking water from private wells contributes to the overall PFAS body burden in Gustavus residents.

Tactical Circuit Training Improves Blood Pressure and Vascular Health More Than Resistance Training.

Exercise training is known to reduce CVD risk factors; however, in tactical populations, like veterans and firefighters, the effects of different forms of exercise such as tactical circuit training (CT) or conventional resistance training (RT) is unclear. Thus, the purpose of this study was to compare changes in various CVD risk measures after 4-week tactical CT or RT programs. Thirty-seven firefighters (20 CT, 17 RT), 35% of whom were veterans, participated. Pre- and post-intervention measures included body fat (BF%), carotid artery intima media thickness (IMT), central and brachial BP, and indices of arterial stiffness (augmentation index, Aix@75), myocardial oxygenation (subendocardial viability ratio, SEVR), and endothelial function (flow-mediated dilation, FMD). Estimation of maximum oxygen consumption (VO2peak) for aerobic fitness, balance, muscular endurance, and strength were also compared. For the clinical laboratory values, there were no between group differences and the only within group change was found in triglyceride levels. Tactical CT lowered triglyceride levels by 24.2% (P < 0.05). Only tactical CT exercise lowered BP. Both brachial (4.6% reduction) and central (4.4 % reduction) systolic and diastolic SBP and DBP decreased with CT (all P ≤ 0.01). After training we found improvements in FMD and SEVR with tactical CT only. Percent FMD increased by 28.7% (P < 0.01) while SEVR increased by 4.4% (P < 0.05) in the tactical CT group. Fitness improved in both cohorts (P < 0.05). These data suggest that 4 weeks of a CT program improves several CVD-risk factors and may be more beneficial.

Improving protection from bioaerosol exposure during postoperative patient interaction in the COVID-19 era, a quality improvement study.

PURPOSE: During patient transport from operating room to post-operative recovery area, anesthesia staff are at increased risk of particle aerosolization from patients despite wearing face shields. Current single-use face shields do not provide anesthesia staff from adequate protection from bioaerosolized particles expired during a patient's cough, particularly during transfer from the operating room to the post-anesthesia recovery unit. In this study, we compare the efficacy of single-use face shield currently available at our institution to a newly designed face shield that provides better protection while still maintaining cost-effectiveness and the ease-of-use of a disposable device. MATERIALS AND METHODS: A patient actor, simulated movements from a patient post-procedure, during transport from operating room to postoperative recovery area. Patterns of exposure of bioaerosolized particles produced from a cough between different face shields was evaluated using fluorescein dye. MAIN RESULTS: More extensive coverage of the lower face, as provided by the Enhanced Protection Face Shield, offers improved droplet protection from bioaerosolized particles emitted from a cough. CONCLUSIONS: Transfer from the operating room to the post-operative recovery unit is a hands-on process and involves managing multiple aspects of patient care physically. Current single-use face shields are convenient and cost-effective, but do not provide adequate protection from droplet aerosolization by patients during transfer. Other masks that provide adequate coverage are costly and are not designed to be single-use. A single-use disposable face shield that offers improved coverage of the lower face provides improved protection for anesthesia staff while maintaining cost-effectiveness, ease-of-use, and infection control.

Adaptive correction of craniofacial defects in pre-metamorphic Xenopus laevis tadpoles involves thyroid hormone-independent tissue remodeling.

Although it is well established that some organisms can regenerate lost structures, the ability to remodel existing malformed structures has been less well studied. Therefore, in this study we examined the ability of pre-metamorphic Xenopus laevis tadpoles to self-correct malformed craniofacial tissues. We found that tadpoles can adaptively improve and normalize abnormal craniofacial morphology caused by numerous developmental perturbations. We then investigated the tissue-level and molecular mechanisms that mediate the self-correction of craniofacial defects in pre-metamorphic X. laevis tadpoles. Our studies revealed that this adaptive response involves morphological changes and the remodeling of cartilage tissue, prior to metamorphosis. RT-qPCR and RNA-seq analysis of gene expression suggests a thyroid hormone-independent endocrine signaling pathway as the potential mechanism responsible for triggering the adaptive and corrective remodeling response in these larvae that involves mmp1 and mmp13 upregulation. Thus, investigating how malformed craniofacial tissues are naturally corrected in X. laevis tadpoles has provided valuable insights into the maintenance and manipulation of craniofacial morphology in a vertebrate system. These insights may help in the development of novel therapies for developmental craniofacial anomalies in humans.

Mechanisms of physiological tissue remodeling in animals: Manipulating tissue, organ, and organism morphology.

Tissue remodeling is broadly defined as the reorganization or restoration of existing tissues. Tissue remodeling processes are responsible for directing the development and maintenance of tissues, organs, and overall morphology of an organism. Therefore, studying the regulatory and mechanistic aspects of tissue remodeling allows one to decipher how tissue structure and function is manipulated in animals. As such, research focused on investigating natural tissue reorganization in animal model organisms has great potential for advancing medical therapies, in conjunction with tissue engineering and regenerative medicine. Here we discuss the molecular and cellular mechanisms responsible for tissue remodeling events that occur across several animal phyla. Notably, this review emphasizes the molecular and cellular mechanisms involved in embryonic and postnatal physiological tissue remodeling events, ranging from metamorphosis to bone remodeling during functional adaptation.

Recovery of the Xenopus laevis heart from ROS-induced stress utilizes conserved pathways of cardiac regeneration.

Urodele amphibians and some fish are capable of regenerating up to a quarter of their heart tissue after cardiac injury. While many anuran amphibians like Xenopus laevis are not capable of such feats, they are able to repair lesser levels of cardiac damage, such as that caused by oxidative stress, to a far greater degree than mammals. Using an optogenetic stress induction model that utilizes the protein KillerRed, we have investigated the extent to which mechanisms of cardiac regeneration are conserved during the restoration of normal heart morphology post oxidative stress in X. laevis tadpoles. We focused particularly on the processes of cardiomyocyte proliferation and dedifferentiation, as well as the pathways that facilitate the regulation of these processes. The cardiac response to KillerRed-induced injury in X. laevis tadpole hearts consists of a phase dominated by indicators of cardiac stress, followed by a repair-like phase with characteristics similar to mechanisms of cardiac regeneration in urodeles and fish. In the latter phase, we found markers associated with partial dedifferentiation and cardiomyocyte proliferation in the injured tadpole heart, which, unlike in regenerating hearts, are not dependent on Notch or retinoic acid signaling. Ultimately, the X. laevis cardiac response to KillerRed-induced oxidative stress shares characteristics with both mammalian and urodele/fish repair mechanisms, but is nonetheless a unique form of recovery, occupying an intermediate place on the spectrum of cardiac regenerative ability. An understanding of how Xenopus repairs cardiac damage can help bridge the gap between mammals and urodeles and contribute to new methods of treating heart disease.

Effects of circuit exercise training on vascular health and blood pressure.

As the global burden of cardiovascular disease (CVD) rises, public health-related interventions aimed at prevention of heart disease have gained medical attention. Clinical research reports that exercise is a protective risk factor associated with CVD and that clinicians need to provide exercise recommendations to patients. Nevertheless, physical inactivity remains a public health problem. In certain populations, like firefighters (FF), increased risk of CVD is especially concerning. The workload FF face is extreme, 50% of line-of-duty deaths (LODD) in FF are cardiac-related, and research on the volunteer FF population is scarce. Government regulations do not require volunteer FF companies to have fitness testing or programming, so exercise intervention studies are necessary to improve the burden of CVD risk in this population. Therefore, this study examined the effects of a 4-week exercise circuit training (CT) intervention on vascular health and fitness in volunteer FF (N = 27) from the Philadelphia PA area compared to a control group of Non-FF (N = 25). Carotid artery intima-media thickness (IMT), brachial artery flow-mediated dilation (FMD), augmentation index, and pulse pressure (PP), brachial and central blood pressure (BP) and fitness were measured pre- and post- intervention. Overall, volunteer FF had more significant improvements (p < 0.05) in vascular health measures (FMD, IMT, and PP). In both groups, we also found that brachial and central BP decreased with exercise. We show that a 4 week CT program can improve vascular structure and function in the volunteer FF population, suggesting that clinicians may be able to reduce or prevent cardiac LODD by exercise.

Bioelectric signaling in regeneration: Mechanisms of ionic controls of growth and form.

The ability to control pattern formation is critical for the both the embryonic development of complex structures as well as for the regeneration/repair of damaged or missing tissues and organs. In addition to chemical gradients and gene regulatory networks, endogenous ion flows are key regulators of cell behavior. Not only do bioelectric cues provide information needed for the initial development of structures, they also enable the robust restoration of normal pattern after injury. In order to expand our basic understanding of morphogenetic processes responsible for the repair of complex anatomy, we need to identify the roles of endogenous voltage gradients, ion flows, and electric fields. In complement to the current focus on molecular genetics, decoding the information transduced by bioelectric cues enhances our knowledge of the dynamic control of growth and pattern formation. Recent advances in science and technology place us in an exciting time to elucidate the interplay between molecular-genetic inputs and important biophysical cues that direct the creation of tissues and organs. Moving forward, these new insights enable additional approaches to direct cell behavior and may result in profound advances in augmentation of regenerative capacity.

HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner.

Laterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels (hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN4) is important for correct left-right patterning. HCN4 channels are present very early in Xenopus embryos. Blocking HCN channels (Ih currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.

Coordinating heart morphogenesis: A novel role for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels during cardiogenesis in Xenopus laevis.

Hyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.

Use of Xenopus Frogs to Study Renal Development/Repair.

The Xenopus genus includes several members of aquatic frogs native to Africa but is perhaps best known for the species Xenopus laevis and Xenopus tropicalis. These species were popularized as model organisms from as early as the 1800s and have been instrumental in expanding several biological fields including cell biology, environmental toxicology, regenerative biology, and developmental biology. In fact, much of what we know about the formation and maturation of the vertebrate renal system has been acquired by examining the intricate genetic and morphological patterns that epitomize nephrogenesis in Xenopus. From these numerous reports, we have learned that the process of kidney development is as unique among organs as it is conserved among vertebrates. While development of most organs involves increases in size at a single location, development of the kidney occurs through a series of three increasingly complex nephric structures that are temporally distinct from one another and which occupy discrete spatial locales within the body. These three renal systems all serve to provide homeostatic, osmoregulatory, and excretory functions in animals. Importantly, the kidneys in amphibians, such as Xenopus, are less complex and more easily accessed than those in mammals, and thus tadpoles and frogs provide useful models for understanding our own kidney development. Several descriptive and mechanistic studies conducted with the Xenopus model system have allowed us to elucidate the cellular and molecular mediators of renal patterning and have also laid the foundation for our current understanding of kidney repair mechanisms in vertebrates. While some species-specific responses to renal injury have been observed, we still recognize the advantage of the Xenopus system due to its distinctive similarity to mammalian wound healing, reparative, and regenerative responses. In addition, the first evidence of renal regeneration in an amphibian system was recently demonstrated in Xenopus laevis. As genetic and molecular tools continue to advance, our appreciation for and utilization of this amphibian model organism can only intensify and will certainly provide ample opportunities to further our understanding of renal development and repair.

Beyond the Mammalian Heart: Fish and Amphibians as a Model for Cardiac Repair and Regeneration.

The epidemic of heart disease, the leading cause of death worldwide, is made worse by the fact that the adult mammalian heart is especially poor at repair. Damage to the mammal heart-such as that caused by myocardial infarction-leads to scarring, resulting in cardiac dysfunction and heart failure. In contrast, the hearts of fish and urodele amphibians are capable of complete regeneration of cardiac tissue from multiple types of damage, with full restoration of functionality. In the last decades, research has revealed a wealth of information on how these animals are able to perform this remarkable feat, and non-mammalian models of heart repair have become a burgeoning new source of data on the morphological, cellular, and molecular processes necessary to heal cardiac damage. In this review we present the major findings from recent research on the underlying mechanisms of fish and amphibian heart regeneration. We also discuss the tools and techniques that have been developed to answer these important questions.

Optogenetic Control of Apoptosis in Targeted Tissues of Xenopus laevis Embryos.

KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies-particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney.

Regeneration of functional pronephric proximal tubules after partial nephrectomy in Xenopus laevis.

BACKGROUND: While the renal system is critical for maintaining homeostatic equilibrium within the body, it is also susceptible to various kinds of damage. Tubule dysfunction in particular contributes to acute renal injury and chronic kidney disease in millions of patients worldwide. Because current treatments are highly invasive and often unavailable, gaining a better understanding of the regenerative capacity of renal structures is vital. Although the effects of various types of acute damage have been previously studied, the ability of the excretory system to repair itself after dramatic tissue loss due to mechanical damage is less well characterized. RESULTS: A novel unilateral nephrectomy technique was developed to excise pronephric proximal tubules from Xenopus laevis tadpoles to study tubule repair after injury. Immunohistochemical detection of protein expression and renal uptake assays demonstrated that X. laevis larvae have the capacity to regenerate functional proximal tubules following resection. CONCLUSIONS: We have validated the renal identity of the restored tubules and demonstrated their ability to functional normally providing the first evidence of regeneration of renal tissue in an amphibian system. Importantly, this tubule restoration occurs by means of a process involving an early apoptotic event and the biphasic expression of the matrix metalloproteinase, Xmmp-9.

Low concentrations of atrazine, glyphosate, 2,4-dichlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis.

Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides (atrazine, 2,4-dichloropheoxyacetic acid (2,4-D), triadimefon, and glyphosate) in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.

Acute atrazine exposure disrupts matrix metalloproteinases and retinoid signaling during organ morphogenesis in Xenopus laevis.

Exposure to the herbicide atrazine disrupts many developmental processes in non-target animals. Atrazine exposure during organ morphogenesis in amphibians results in dramatic malformations; the mechanism by which this happens has not been described. We have taken a candidate gene approach to explore two possible mechanisms by which acute atrazine exposure causes extensive malformations in several tissues in Xenopus laevis tadpoles. Using a static renewal system, we exposed tadpoles to atrazine for 6-48 h during organ morphogenesis (Nieuwkoop and Faber stage 42). We observed degradation of cranial cartilage and differentiated muscle in the head, gut and somites of exposed tadpoles. Additionally, transcript levels of matrix metalloproteinases (MMPs), specifically both MMP9TH and MMP18, increased in atrazine-exposed tadpoles in a dose-response test, and MMP18 increased as early as 6 h after exposure began. Gelatinase MMP activity was also altered by atrazine exposure, indicating that atrazine disrupts gene function at the level of transcription and protein activity. Furthermore, transcript levels of the enzyme Xcyp26, an enzyme in the retinoic acid signaling pathway, significantly decreased in the intestines of tadpoles exposed to 10 or 35 mg l(-1) atrazine for 48 h. Our results suggest two mechanisms by which atrazine can disrupt tissue morphogenesis: through misregulation of MMPs that are critical in extracellular matrix remodeling throughout development and the disruption of retinoic acid signaling. This study begins to describe conserved vertebrate developmental processes that are disrupted by atrazine exposure.

Bioelectric controls of cell proliferation: ion channels, membrane voltage and the cell cycle.

All cells possess long-term, steady-state voltage gradients across the plasma membrane. These transmembrane potentials arise from the combined activity of numerous ion channels, pumps and gap junction complexes. Increasing data from molecular physiology now reveal that the role of changes in membrane voltage controls, and is in turn controlled by, progression through the cell cycle. We review recent functional data on the regulation of mitosis by bioelectric signals, and the function of membrane voltage and specific potassium, sodium and chloride ion channels in the proliferation of embryonic, somatic and neoplastic cells. Its unique properties place this powerful, well-conserved, but still poorly-understood signaling system at the center of the coordinated cellular interactions required for complex pattern formation. Moreover, disregulation of ion channel expression and function is increasingly observed to be not only a useful marker but likely a functional element in oncogenesis. New advances in genomics and the development of in vivo biophysical techniques suggest exciting opportunities for molecular medicine, bioengineering and regenerative approaches to human health.

Coordinating the timing of cardiac precursor development during gastrulation: a new role for Notch signaling.

Notch signaling has been shown to mediate a wide array of cell fate decisions during development. While previous work has demonstrated that Notch signaling plays an important role in regulating cardiac differentiation and morphogenesis, an earlier role during cardiac field formation has not yet been fully characterized. Previously, our lab demonstrated that perturbations in Notch signaling beginning at the onset of gastrulation affect the subdivision of germ layers. However due to the potential additive effects of misregulating Notch signaling over multiple stages of development, it was not possible to distinguish a specific role for this pathway during heart field specification. Here, we developed an innovative approach that takes advantage of temporally inducible constructs to isolate our manipulations to specific windows of development. In particular, we focused our studies on some of the earliest stages of cardiogenesis when heart field specification occurs. Our findings demonstrate a novel role for Notch signaling during the prepatterning of the cardiac mesoderm. Specifically, once relieved of aberrantly activated Notch signaling following gastrulation, cardiac precursors retain the ability to express markers of the cardiac field. Conversely, downregulating Notch signaling in cells fated to become heart tissue results in the induction of cardiac field genes in gastrula embryos. Finally, we provide evidence suggesting that this new role for Notch signaling is mediated at least in part via the Notch effector protein, Esr9 and the transcription factor GATA4. Taken together, these findings provide strong evidence for a novel role for Notch signaling in regulating the timing of heart field specification during early cardiogenesis.