Xenopus: Experimental access to cardiovascular development, regeneration discovery, and cardiovascular heart-defect modeling

Xenopus has been used to study a wide array of developmental processes, benefiting from vast quantities of relatively large, externally developing eggs. Xenopus is particularly amenable to examining the cardiac system because many of the developmental processes and genes involved in cardiac specification, differentiation, and growth are conserved between Xenopus and human and have been characterized in detail. Furthermore, compared with other higher vertebrate models, Xenopus embryos can survive longer without a properly functioning heart or circulatory system, enabling investigation of later consequences of early embryological manipulations. This biology is complemented by experimental technology, such as embryonic explants to study the heart, microinjection of overexpression constructs, and, most recently, the generation of genetic mutations through gene-editing technologies. Recent investigations highlight Xenopus as a powerful experimental system for studying injury/repair and regeneration and for congenital heart disease (CHD) modeling, which reinforces why this model system remains ideal for studying heart development

Proteomic-based approaches to cardiac development and disease

Congenital malformations, or structural birth defects, are now the leading cause of infant mortality in the United States and Europe (Dolk et al., 2010; Heron et al., 2009). Of the congenital malformations, congenital heart disease (CHD) is the most common (Dolk et al., 2010; Heron et al., 2009). Thus, a molecular understanding of heart development is an essential goal for improving clinical approaches to CHD. However, CHDs are commonly a result of genetic defects that manifest themselves in a spatial and temporal manner during the early stages of embryogenesis, leaving them mostly intractable to mass spectrometry-based analysis. Here, we describe the technologies and advancements in the field of mass spectrometry over the past few years that have begun to provide insights into the molecular and cellular basis of CHD and prospects for these types of approaches in the future

The T-box family

Transcription factors of the T-box family are required both for early cell-fate decisions, such as those necessary for formation of the basic vertebrate body plan, and for differentiation and organogenesis. When mutated, T-box genes give dramatic phenotypes in mouse and zebrafish, and they have been implicated both in fundamentals of limb patterning and in a number of human congenital malformations such as Holt-Oram, ulnar-mammary and DiGeorge syndromes, as well as being amplified in a subset of cancers. Genes encoding members of the T-box family have recently been shown to comprise approximately 0.1% of genomes as diverse as those of nematodes and humans and have been identified in a wide variety of animals from ctenophores (comb jellies) to mammals; they are, however, completely absent from genomes from other organisms (such as the model plant Arabidopsis thaliana)

INTACT proteomics in Xenopus

Analysis of the molecular mechanisms driving cell specification, differentiation, and other cellular processes can be difficult due to the heterogeneity of tissues and organs. Therefore, it is critical to isolate pure cell populations in order to properly assess the function of certain cell types in the context of a tissue. This protocol describes use of the INTACT (isolation of nuclei tagged in specific cell types) method in Xenopus, followed by proteomics analysis of nuclear protein complexes. The INTACT protocol utilizes two transgenes: (1) a three-part nuclear targeting fusion (NTF) consisting of a nuclear envelope protein (Nup35) that targets the NTF to the nuclear membrane, an enhanced green fluorescent protein (EGFP) cassette for NTF visualization in live animals, and a biotin ligase receptor protein (BLRP) that provides a substrate for the biotinylation of the NTF, and (2) the E. coli ligase BirA (which biotinylates the NTF) tagged to mCherry (for visualization). Either or both transgenes are driven by a tissue-specific promoter, making this protocol easily adaptable to proteomics analyses of immunoprecipitated complexes from INTACT-isolated nuclei of multiple tissue types to determine the composition of protein complexes in pure cell populations

CHD4 and the NuRD complex directly control cardiac sarcomere formation

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development

Initiating Events in Direct Cardiomyocyte Reprogramming

Direct reprogramming of fibroblasts into cardiomyocyte-like cells (iCM) holds great potential for heart regeneration and disease modeling and may lead to future therapeutic applications. Currently, application of this technology is limited by our lack of understanding of the molecular mechanisms that drive direct iCM reprogramming. Using a quantitative mass spectrometry-based proteomic approach, we identified the temporal global changes in protein abundance that occur during initial phases of iCM reprogramming. Collectively, our results show systematic and temporally distinct alterations in levels of specific functional classes of proteins during the initiating steps of reprogramming including extracellular matrix proteins, translation factors, and chromatin-binding proteins. We have constructed protein relational networks associated with the initial transition of a fibroblast into an iCM. These findings demonstrate the presence of an orchestrated series of temporal steps associated with dynamic changes in protein abundance in a defined group of protein pathways during the initiating events of direct reprogramming. Translational application of direct reprogramming into cardiomyocyte-like cells is limited by our understanding of the molecular mechanisms underlying initiating steps. Sauls et al. define an orchestrated series of temporal changes in the abundance of proteins and protein pathways during early stages of cardiac reprogramming

Conservation and divergence of protein pathways in the vertebrate heart

Heart disease is the leading cause of death in the western world. Attaining a mechanistic understanding of human heart development and homeostasis and the molecular basis of associated disease states relies on the use of animal models. Here, we present the cardiac proteomes of 4 model vertebrates with dual circulatory systems: the pig (Sus scrofa), the mouse (Mus musculus), and 2 frogs (Xenopus laevis and Xenopus tropicalis). Determination of which proteins and protein pathways are conserved and which have diverged within these species will aid in our ability to choose the appropriate models for determining protein function and to model human disease. We uncover mammalian- and amphibian-specific, as well as species-specific, enriched proteins and protein pathways. Among these, we find and validate an enrichment in cell-cycle–associated proteins within Xenopus laevis. To further investigate functional units within cardiac proteomes, we develop a computational approach to profile the abundance of protein complexes across species. Finally, we demonstrate the utility of these data sets for predicting appropriate model systems for studying given cardiac conditions by testing the role of Kielin/chordin-like protein (Kcp), a protein found as enriched in frog hearts compared to mammals. We establish that germ-line mutations in Kcp in Xenopus lead to valve defects and, ultimately, cardiac failure and death. Thus, integrating these findings with data on proteins responsible for cardiac disease should lead to the development of refined, species-specific models for protein function and disease states

Cardiac proteomics reveals sex chromosome-dependent differences between males and females that arise prior to gonad formation

Sex disparities in cardiac homeostasis and heart disease are well documented, with differences attributed to actions of sex hormones. However, studies have indicated sex chromosomes act outside of the gonads to function without mediation by gonadal hormones. Here, we performed transcriptional and proteomics profiling to define differences between male and female mouse hearts. We demonstrate, contrary to current dogma, cardiac sex disparities are controlled not only by sex hormones but also through a sex-chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) models, we find the sex-chromosome pathway is established by X-linked gene dosage. We demonstrate cardiac sex disparities occur at the earliest stages of heart formation, a period before gonad formation. Using these datasets, we identify and define a role for alpha-1B-glycoprotein (A1BG), showing loss of A1BG leads to cardiac defects in females, but not males. These studies provide resources for studying sex-biased cardiac disease states

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life