Pharyngeal Mesoderm Research Articles

A new Prdm1-Cre line is suitable for studying the second heart field development

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

Supraclavicular brown adipocytes originate from Tbx1+ myoprogenitors.

Brown adipose tissue (BAT) dissipates energy as heat, contributing to temperature control, energy expenditure, and systemic homeostasis. In adult humans, BAT mainly exists in supraclavicular areas and its prevalence is associated with cardiometabolic health. However, the developmental origin of supraclavicular BAT remains unknown. Here, using genetic cell marking in mice, we demonstrate that supraclavicular brown adipocytes do not develop from the Pax3+/Myf5+ epaxial dermomyotome that gives rises to interscapular BAT (iBAT). Instead, the Tbx1+ lineage that specifies the pharyngeal mesoderm marks the majority of supraclavicular brown adipocytes. Tbx1Cre-mediated ablation of peroxisome proliferator-activated receptor gamma (PPARγ) or PR/SET Domain 16 (PRDM16), components of the transcriptional complex for brown fat determination, leads to supraclavicular BAT paucity or dysfunction, thus rendering mice more sensitive to cold exposure. Moreover, human deep neck BAT expresses higher levels of the TBX1 gene than subcutaneous neck white adipocytes. Taken together, our observations reveal location-specific developmental origins of BAT depots and call attention to Tbx1+ lineage cells when investigating human relevant supraclavicular BAT.

Exposure to the aryl hydrocarbon receptor agonist dioxin disrupts formation of the muscle, nerves, and vasculature in the developing jaw

Human exposure to environmental pollutants can disrupt embryonic development and impact juvenile and adult health outcomes by adversely affecting cell and organ function. Notwithstanding, environmental contamination continues to increase due to industrial development, insufficient regulations, and the mobilization of pollutants as a result of extreme weather events. Dioxins are a class of structurally related persistent organic pollutants that are highly toxic, carcinogenic, and teratogenic. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin compound and has been shown to induce toxic effects in developing organisms by activating the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor targeted by multiple persistent organic pollutants. Contaminant-induced AHR activation results in malformations of the craniofacial cartilages and neurocranium; however, the mechanisms mediating these phenotypes are not well understood. In this study, we utilized the optically transparent zebrafish model to elucidate novel cellular targets and potential transcriptional targets underlying TCDD-induced craniofacial malformations. To this end, we exposed zebrafish embryos at 4 h post fertilization to TCDD and employed a mixed-methods approach utilizing immunohistochemistry staining, transgenic reporter lines, fixed and in vivo confocal imaging, and timelapse microscopy to determine the targets mediating TCDD-induced craniofacial phenotypes. Our data indicate that embryonic TCDD exposure reduced jaw and pharyngeal arch Sox10+ chondrocytes and Tcf21+ pharyngeal mesoderm progenitors. Exposure to TCDD correspondingly led to a reduction in collagen type II deposition in Sox10+ domains. Embryonic TCDD exposure impaired development of tissues derived from or guided by Tcf21+ progenitors, namely: nerves, muscle, and vasculature. Specifically, TCDD exposure disrupted development of the hyoid and mandibular arch muscles, decreased neural innervation of the jaw, resulted in compression of cranial nerves V and VII, and led to jaw vasculature malformations. Collectively, these findings reveal novel structural targets and potential transcriptional targets of TCDD-induced toxicity, showcasing how contaminant exposures lead to congenital craniofacial malformations.

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

An Appreciation of Anatomy in the Molecular World.

Robert H. Anderson is one of the most important and accomplished cardiac anatomists of the last decades, having made major contributions to our understanding of the anatomy of normal hearts and the pathologies of acquired and congenital heart diseases. While cardiac anatomy as a research discipline has become largely subservient to molecular biology, anatomists like Professor Anderson demonstrate anatomy has much to offer. Here, we provide cases of early anatomical insights on the heart that were rediscovered, and expanded on, by molecular techniques: migration of neural crest cells to the heart was deduced from histological observations (1908) and independently shown again with experimental interventions; pharyngeal mesoderm is added to the embryonic heart (1973) in what is now defined as the molecularly distinguishable second heart field; chambers develop from the heart tube as regional pouches in what is now considered the ballooning model by the molecular identification of regional differentiation and proliferation. The anatomical discovery of the conduction system by Purkinje, His, Tawara, Keith, and Flack is a special case because the main findings were never neglected in later molecular studies. Professor Anderson has successfully demonstrated that sound knowledge of anatomy is indispensable for proper understanding of cardiac development.

Generation of craniofacial myogenic progenitor cells from human induced pluripotent stem cells for skeletal muscle tissue regeneration

Craniofacial skeletal muscle is composed of approximately 60 muscles, which have critical functions including food uptake, eye movements and facial expressions. Although craniofacial muscles have significantly different embryonic origin, most current skeletal muscle differentiation protocols using human induced pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscle developmental pathways. Since the lack of a protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we have developed an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human iPSCs by mimicking key signaling pathways during craniofacial embryonic myogenesis. At each different stage, human iPSC-derived cMPCs mirror the transcription factor expression profiles seen in their counterparts during embryo development. After the bi-potential cranial pharyngeal mesoderm is established, cells are committed to cranial skeletal muscle lineages with inhibition of cardiac lineages and are purified by flow cytometry. Furthermore, identities of iPSC-derived cMPCs are verified with human primary myoblasts from craniofacial muscles using RNA sequencing. These data suggest that our new method could provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also abundant and reliable cellular resources for tissue engineering to support craniofacial reconstruction surgery.

P63 Mutation Impacts the Structure of Cardiopharyngeal Field Derived Muscles in Mice

The cardiopharyngeal field (CPF) includes anterior lateral mesoderm of the first heart field and the contiguous pharyngeal mesoderm. The latter gives rise to second‐heart‐field‐derived heart regions and branchiomeric head muscles. Neural crest cells (NCCs) surround and infiltrate mesoderm cells, forming connective tissue of head and heart musculature. NCCs are also involved in the septation of the cardiac outflow tract, the patterning of pharyngeal arch arteries, and in the proper development of attachments of branchiomeric muscles to skull, jaw, hyoid bone, and laryngeal cartilages.The p63 (aka Trp63) transcription factor is essential for the differentiation of epithelial tissue (ectoderm and endoderm) during embryogenesis. To form the correct amount of mature ectodermal, mesodermal, and NCC populations and to control the differentiation of the CPF, p63 interacts with downstream transcription factors, such as Tbx1, Isl1, and nkx2.5. We analyzed how the loss of p63 expression in p63−/− mice affected the mesodermal derivatives of the CPF. We hypothesized that the loss of p63 decreases the number of mesodermal cells differentiating from the CPF, causing a smaller mesodermal cell population in the CPF. We expected to see smaller CPF derived muscles in the p63 mutant mice, as well as deficient connective tissue in head and heart musculature as well as weakened attachments of craniofacial muscles.To determine if and when certain muscular defects appear, we performed muscle antibody staining and dissected wildtype littermates and p63−/− mice (B6.129S7‐Trp63tm2Brd/J strain) aged embryonic day (E) 12–18. We have collected minimum of five specimens for each age. We studied craniofacial, laryngeal, as well as cardiac musculature by analyzing the antibody stained specimens under the microscope and by performing microdissections under a stereomicroscope. We used 3D computed tomography scan images (taken with a uCT scanner, Skyscan 1172) to compare the craniofacial skeletons between wildtype and mutant mice.In p63−/− mice, branchiomeric muscles such as the masseter, buccinator, and nasolabialis were properly attached to bony elements but were smaller than what was seen in the wildtype mice. The p63−/− head is flexed and the chin is closer to the chest than in the wildtype specimens: this posture could be caused by the shortened and less dense sternocleidomastoid muscle in the p63−/− mice. Corresponding with previous reports, our p63 −/− mice also showed several malformations (e.g., poorly developed limbs, cleft palate, cardiac abnormalities) stereotypical of this mouse mutant and which are also seen in humans with p63 mutations such as in ADULT (acro‐dermatoungual‐lacrimal‐tooth) and EEC (Ectrodactyly‐Ectodermal Dysplasia‐Cleft Lip /Palate) syndromes. Despite that p63 expression is restricted to epithelium, the described defects reveal that p63 expression is important to much earlier, and shared developmental processes, notably NCC proliferation and differentiation, that do impact tissues derived from mesoderm as well as from epithelium. Analyzing the influence of p63 on CPF differentiation will contribute to a better understanding of regulatory mechanisms during head and heart development including the role of these mechanisms in human congenital syndromes.

Pharyngeal pouches provide a niche microenvironment for arch artery progenitor specification

ABSTRACTThe paired pharyngeal arch arteries (PAAs) are transient blood vessels connecting the heart with the dorsal aorta during embryogenesis. Although PAA malformations often occur along with pharyngeal pouch defects, the functional interaction between these adjacent tissues remains largely unclear. Here, we report that pharyngeal pouches are essential for PAA progenitor specification in zebrafish embryos. We reveal that the segmentation of pharyngeal pouches coincides spatiotemporally with the emergence of PAA progenitor clusters. These pouches physically associate with pharyngeal mesoderm in discrete regions and provide a niche microenvironment for PAA progenitor commitment by expressing BMP proteins. Specifically, pouch-derived BMP2a and BMP5 are the primary niche cues responsible for activating the BMP/Smad pathway in pharyngeal mesoderm, thereby promoting progenitor specification. In addition, BMP2a and BMP5 play an inductive function in the expression of the cloche gene npas4l in PAA progenitors. cloche mutants exhibit a striking failure to specify PAA progenitors and display ectopic expression of head muscle markers in the pharyngeal mesoderm. Therefore, our results support a crucial role for pharyngeal pouches in establishing a progenitor niche for PAA morphogenesis via BMP2a/5 expression.

HIF-1α is required for development of the sympathetic nervous system

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.

A single-cell transcriptional roadmap for cardiopharyngeal fate diversification.

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programs and mechanisms underlying cardiopharyngeal multipotency and heart vs. head muscle fate choices remain elusive. Here, we used single cell genomics in the simple chordate model Ciona, to reconstruct developmental trajectories forming first and second heart lineages, and pharyngeal muscle precursors, and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signaling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac program, shared by the first and second lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage program, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.

Isl1-expressing non-venous cell lineage contributes to cardiac lymphatic vessel development

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1+ lineage-specific deletion of Prox1 resulted in disrupted LYVE1+ vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3+ and/or Prox1+ cells that overlapped with the Isl1+ pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis.

Day 0–28 of Human Embryonic Development: Importance of Studying Cell and Tissue Interactions in Head and Heart Development

All vertebrates start their development from a zygote and the processes from this cell to a complex organism are often complicated and include many gene regulatory networks, cell migrations, cell differentiations, communication between cells and between tissues, etc. Earliest mis‐regulations in those processes lead almost always to the demise of the developing embryo – often before the women even realizes she is pregnant. This is because the most exciting and complex processes related to body plan formation happen during the first 8 weeks of embryonic development. Even more fascinating is that at the end of the first months (28 days after fertilization) the head and heart region are clearly distinguishable: the heart is beating, u‐shaped and atria and ventricles are aligned, the pharyngeal arches are present, as are eye and ear anlagen (placodes), in the brain several regions are distinct, including forebrain, midbrain, and hindbrain. There are many remarkable research groups all over the world studying one or the other system (brain, cardiovascular, craniofacial), but only few groups study the interactions between all tissues (ectoderm, mesoderm, endoderm – neural crest as special tissue derived from ectoderm). However, understanding precisely those interactions during earliest and later development will help us to understand human syndromes with the same underlying genetic defect but that present with a huge variety of phenotypes.For example, the DiGeorge Syndrome is caused by a deletion of a region at 22q11.2 that contains 30–40 genes. Due to its involvement of several system it is also called CATCH22 – Cardiac anomalies, Abnormal facies, Thymic hypoplasia, Cleft palate, Hypocalcemia. Each of those abnormalities could be caused by the involvement of cranial neural crest cells during embryonic development, even the heart anomalies as cardiac neural crest cells are part of the posterior population of cranial neural crest cells. Also, in the region of the 22q11.2 is the Tbx1 gene that was shown to be essential during earliest cardiopharyngeal mesoderm development and differentiation. The cardiopharyngeal field is a progenitor field that gives rise to the first heart field and the pharyngeal mesoderm. The latter will differentiate into the second heart field derived cardiac musculature and the branchiomeric (head) muscles. Yet, neither our knowledge of neural crest cell involvement nor our understanding of cardiopharyngeal mesoderm development, fully explains the variety of symptoms seen in DiGeorge Syndrome patients.Therefore, a detailed summary of what is currently known about the first 28 days of head and heart development and the tissue interactions will help us to understand complex syndromes. Furthermore, it will be possible to identify regions that need more research focus. Last, but not least, a better understanding of our own developmental processes also provides insights into evolutionary processes and studying animal developmental processes further contributes to a deepened knowledge of the evolutionary development of our own body. Here, I will present a summary of the first 28 days of head and heart embryonic development in humans and highlight regions of research that next generation scientists might be interested in.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of actin cytoskeleton in regulating the deployment process of mouse cardiac second heart field progenitor cells

The vertebrate heart tube originates from cardiogenic mesodermal cells in the early embryo, and subsequently elongates by progressive addition of second heart field (SHF) progenitor cells from adjacent pharyngeal mesoderm and splanchnic mesoderm. Insufficient addition of SHF cells to the heart tube causes the failure of maximal elongation of the heart tube, which results in a series of developmental defects including the most common congenital birth defects, such as right ventricular dysplasia and outflow tract septation, and alignment anomalies. SHF cells form an atypical, apicobasally polarized epithelium which is characterized by apical monocilia, and dynamic actin-rich basal filopodia. In this review, we summarize recent research progresses of actin cytoskeleton in the deployment process of mouse SHF progenitor cells, and reveal the significance of actin cytoskeleton in SHF development, especially in the deployment of SHF cells to the outflow tract, to provide theoretical reference for elucidating and understanding the biological characteristics of SHF deployment.

Nkx2\u20135 Second Heart Field Target Gene Ccdc117 Regulates DNA Metabolism and Proliferation

The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5−/− null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117′s interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.

Wnt/\u03b2-catenin-mediated signaling re-activates proliferation of matured cardiomyocytes

BackgroundThe Wnt/β-catenin signaling pathway plays an important role in the development of second heart field (SHF Isl1+) that gives rise to the anterior heart field (AHF) cardiac progenitor cells (CPCs) for the formation of the right ventricle, outflow tract (OFT), and a portion of the inflow tract (IFT). During early cardiogenesis, these AHF CPCs reside within the pharyngeal mesoderm (PM) that provides a microenvironment for them to receive signals that direct their cell fates. Here, N-cadherin, which is weakly expressed by CPCs, plays a significant role by promoting the adhesion of CPCs within the AHF, regulating β-catenin levels in the cytoplasm to maintain high Wnt signaling and cardioproliferation while also preventing the premature differentiation of CPCs. On the contrary, strong expression of N-cadherin observed throughout matured myocardium is associated with downregulation of Wnt signaling due to β-catenin sequestration at the cell membrane, inhibiting cardioproliferation. As such, upregulation of Wnt signaling pathway to enhance cardiac tissue proliferation in mature cardiomyocytes can be explored as an interesting avenue for regenerative treatment to patients who have suffered from myocardial infarction.MethodsTo investigate if Wnt signaling is able to enhance cellular proliferation of matured cardiomyocytes, we treated cardiomyocytes isolated from adult mouse heart and both murine and human ES cell-derived matured cardiomyocytes with N-cadherin antibody or CHIR99021 GSK inhibitor in an attempt to increase levels of cytoplasmic β-catenin. Immunostaining, western blot, and quantitative PCR for cell proliferation markers, cell cycling markers, and Wnt signaling pathway markers were used to quantitate re-activation of cardioproliferation and Wnt signaling.ResultsN-cadherin antibody treatment releases sequestered β-catenin at N-cadherin-based adherens junction, resulting in an increased pool of cytoplasmic β-catenin, similar in effect to CHIR99021 GSK inhibitor treatment. Both treatments therefore upregulate Wnt signaling successfully and result in significant increases in matured cardiomyocyte proliferation.ConclusionAlthough both N-cadherin antibody and CHIR99021 treatment resulted in increased Wnt signaling and cardioproliferation, CHIR99021 was found to be the more effective treatment method for human ES cell-derived cardiomyocytes. Therefore, we propose that CHIR99021 could be a potential therapeutic option for myocardial infarction patients in need of regeneration of cardiac tissue.

T-box genes and retinoic acid signaling regulate the segregation of arterial and venous pole progenitor cells in the murine second heart field.

The arterial and venous poles of the mammalian heart are hotspots of congenital heart defects (CHD) such as those observed in 22q11.2 deletion (or DiGeorge) and Holt-Oram syndromes. These regions of the heart are derived from late differentiating cardiac progenitor cells of the Second Heart Field (SHF) located in pharyngeal mesoderm contiguous with the elongating heart tube. The T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion syndrome gene, regulates SHF addition to both cardiac poles from a common progenitor population. Despite the significance of this cellular addition the mechanisms regulating the deployment of common progenitor cells to alternate cardiac poles remain poorly understood. Here we demonstrate that Tbx5, mutated in Holt-Oram syndrome and essential for venous pole development, is activated in Tbx1 expressing cells in the posterior region of the SHF at early stages of heart tube elongation. A subset of the SHF transcriptional program, including Tbx1 expression, is subsequently downregulated in Tbx5 expressing cells, generating a transcriptional boundary between Tbx1-positive arterial pole and Tbx5-positive venous pole progenitor cell populations. We show that normal downregulation of the definitive arterial pole progenitor cell program in the posterior SHF is dependent on both Tbx1 and Tbx5. Furthermore, retinoic acid (RA) signaling is required for Tbx5 activation in Tbx1-positive cells and blocking RA signaling at the time of Tbx5 activation results in atrioventricular septal defects at fetal stages. Our results reveal sequential steps of cardiac progenitor cell patterning and provide mechanistic insights into the origin of common forms of CHD.

Gene Regulatory Network and Tissue Interactions Important for Head and Heart Development and Evolution

The evolution of both the head and heart are fascinating subjects to explore. Usually, researcher focus on either heart or head and study their development, anatomy, and/or evolution across vertebrates or at smaller taxonomic scales (genera or species). However, the development and evolution of head and heart are tightly linked. The most recent example is the description of the cardiopharyngeal field, which is an area that contains the anterior lateral mesoderm of the first heart field and the adjacent pharyngeal mesoderm that differentiates into branchiomeric muscles and the second heart field derived heart muscle tissue. Simplified, the cardiopharyngeal mesoderm gives rise to head, neck, and heart musculature. Another example are neural crest cells, which are essential for proper connective tissue development of most – if not all branchiomeric muscles. They also give rise to skeletal structures onto which those muscles attach. Furthermore, neural crest cells influence the heart development, as the separation of the outflow tract into pulmonary trunk and aortic arch. Interestingly, the cardiac neural crest is the caudal part of the cranial neural crest. Similarly, endoderm and its proper communication with surrounding mesenchyme plays an important role in head and heart development. All tissues listed here influence each other and complex gene regulatory networks across all layers ensure the differentiation of the head and heart structures.Most of the knowledge we have today regarding those interactions and gene regulatory networks in and between those tissues are from studies of a few model organisms (Ciona, zebrafish, Xenopus, chick, mouse). Recently more species are included in large scale comparative studies. However, rarely is more than one system analyzed per research group. Research that focusses on the interaction between tissues are still rare – mostly because of their complexity and resources needed. But considering our own species, Homo sapiens, we can observe many congenital defects that simultaneously affect craniofacial and cardiac structures. The same is true for many mutant lines for the model species named above. Taking already available specimens and studying the effects across the tissues of the head, neck, and heart structures will help us understanding common patterns. Those can then be traced through the gene regulatory networks and even reveal new links or new candidate genes participating in those networks.One example is Tbx1, which is a marker for pharyngeal mesoderm that includes the second heart field and branchiomeric mesoderm. A microdeletion in the gene is known to lead to DiGeorge syndrome with specific craniofacial and cardiac anomalies. However, Tbx1 is expressed in all embryonic tissues and patients will show a variety of anomalies not only associated with the cardiopharyngeal mesoderm, as development of brain, thyroid and parathyroid glands, kidneys, etc. are also impacted.Support or Funding InformationJMZ is supported by a Howard University College of Medicine pilot study award (BFPSAP: U100217).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of Nkx2.5 and Isl1 in the cardiopharyngeal mesoderm of developing Xenopus embryos

The cardiopharyngeal field (CPF) is a mesodermal field that gives rise to the first (FHF) and second heart field (SHF)‐derived myocardium and the branchiomeric musculature. Isl1 and Nkx2.5 are transcription factors expressed in the CPF. Both genes are crucial for the specification of the myocardial cells derived from the SHF and for the further development of the heart in vertebrates. The importance of these genes is supported by the fact that perturbations in their expression lead to severe heart malformations and often result in embryonic death. In order to better understand the role of these two genes and their impact on the head and heart development, we analysed their expression in Xenopus laevis, which is potentially a good model to study the CPF and craniofacial defects in terms of evolutionary developmental medicine. In contrast to basal vertebrates, such as the urochordate Ciona, Xenopus' heart has an advanced morphology with several heart chambers and in‐ and outflow tracts. Concurrently, Xenopus is easy to rear in laboratory conditions. There are various genetic tools available to work with this species (e.g., TALENs, CRISPR/Cas9, ZFNs) and there are certain advantages for the study of embryology (e.g., external fertilization and large size of eggs) that favour this species over the mouse and chicken models.Whole‐mount in situ hybridization of embryos revealed that during early stages Isl1 and Nkx2.5 are co‐expressed in cardiac progenitor cells localized near the cement gland. Isl1 was also present in the stomodeum region and other cell populations; e.g., cephalic placodes and their associated ganglia or cranial paraxial mesoderm. At stage 25 (Nieuwkoop and Faber), Nkx2.5 was expressed in the FHF, SHF, and anterior endoderm. Later, it nearly separated from the Isl1 expression and became restricted to the heart tube, derived from the FHF. Isl1 was mainly expressed in the pharyngeal arch mesoderm adjacent to the heart and in the dorsolateral heart domain. At late stages, Nkx2.5 could be detected throughout the myocardium, while Isl1 was present in the foregut and cephalic ganglia. In spite of minor differences, our results conform to early studies in X. laevis. The comparison with expression patterns in mouse and chicken shows that Xenopus is indeed a good organism to study the CPF derivatives, like the myocardium and head musculature. Therefore, Xenopus can be further extensively used for developmental, evolutionary, and medical studies of the normal and abnormal head and heart myogenesis.Support or Funding InformationJMZ is supported by a Howard University College of Medicine pilot study award (BFPSAP: U100217).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of T-box gene function in patterning second heart field progenitor cells

The heart is derived from two sources of progenitor cells: the first heart field giving rise to the left ventricle and atria, and the Second Heart Field (SHF) that progressively contributes to the right ventricle, outflow tract and parts of the atria. Perturbation of SHF cell addition leads to a spectrum of congenital heart defects, exemplified by conotruncal and atrioventricular septal defects observed in mouse embryos lacking the T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion (DiGeorge) syndrome candidate gene. Tbx1 is expressed in the SHF where it regulates proliferation, differentiation and epithelial properties. Tbx1 is also required for the segregation of distinct anterior and posterior subpopulations of the SHF giving rise to arterial and venous pole myocardium, respectively. Here we show using genetic lineage tracing and immunofluorescence labeling that a second T-box gene Tbx5 , implicated in Holt-Oram syndrome, is activated in Tbx1-positive pharyngeal mesoderm in response to retinoic acid signaling at the onset of progenitor cell segregation. Loss of retinoic acid signaling results in failure to establish the posterior SHF domain and atrioventricular septal defects. SHF cells coexpressing Tbx1 and Tbx5 subsequently downregulate Tbx1 expression, in a Tbx1-dependent manner, leading to the emergence of a transcriptional boundary between anterior and posterior SHF domains. Analysis of Tbx5 lineage and conditional loss of function implicate de novo activation of Tbx5 in boundary formation. Our results provide new insights into progenitor cell patterning during SHF deployment and the origins of common forms of congenital heart defects.

Regulation and morphogenesis of pharyngeal mesoderm derived muscles

Regulation and morphogenesis of pharyngeal mesoderm derived muscles