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.

Quantitative analyses of adiposity dynamics in zebrafish.

Adipose tissues often exhibit subtle, quantitative differences between individuals, leading to a graded series of adiposity phenotypes at the population level. Robust, quantitative analyses are vital for studying these differences. In this Commentary we highlight two articles from our lab that employ sensitive new methods in zebrafish capable of delineating complex and quantitative adiposity phenotypes. In the first article, we utilized in vivo imaging to systematically quantify zebrafish adipose tissues. We identified 34 regionally distinct zebrafish adipose tissues and developed statistical models to predict the size and variance of each adipose tissue over the course of zebrafish growth. We then employed these models to identify effects of strain and diet on adipose tissue growth. In the second article, we employed deep phenotyping to study complex disease-related adiposity traits. Using this methodology, we identified that adipose tissues have unique capacities to re-deposit lipid following food restriction and re-feeding. These distinct re-deposition potentials led to widespread fat distribution changes following re-feeding. We discuss how these novel findings may provide relevance to health conditions such as anorexia nervosa. Together, the strategies described in these two articles can be used as unbiased and quantitative methods to uncover new relationships between genotype, diet and adiposity.

Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance.

Hypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.

Adipose morphology and metabolic disease.

Adipose morphology is defined as the number and size distribution of adipocytes (fat cells) within adipose tissue. Adipose tissue with fewer but larger adipocytes is said to have a 'hypertrophic' morphology, whereas adipose with many adipocytes of a smaller size is said to have a 'hyperplastic' morphology. Hypertrophic adipose morphology is positively associated with insulin resistance, diabetes and cardiovascular disease. By contrast, hyperplastic morphology is associated with improved metabolic parameters. These phenotypic associations suggest that adipose morphology influences risk of cardiometabolic disease. Intriguingly, monozygotic twin studies have determined that adipose morphology is in part determined genetically. Therefore, identifying the genetic regulation of adipose morphology may help us to predict, prevent and ameliorate insulin resistance and associated metabolic diseases. Here, we review the current literature regarding adipose morphology in relation to: (1) metabolic and medical implications; (2) the methods used to assess adipose morphology; and (3) transcriptional differences between morphologies. We further highlight three mechanisms that have been hypothesized to promote adipocyte hypertrophy and thus to regulate adipose morphology.

The Role of Peroxisome Proliferator-Activated Receptor Gamma (PPARG) in Adipogenesis: Applying Knowledge from the Fish Aquaculture Industry to Biomedical Research.

The tropical freshwater zebrafish has recently emerged as a valuable model organism for the study of adipose tissue biology and obesity-related disease. The strengths of the zebrafish model system are its wealth of genetic mutants, transgenic tools, and amenability to high-resolution imaging of cell dynamics within live animals. However, zebrafish adipose research is at a nascent stage and many gaps exist in our understanding of zebrafish adipose physiology and metabolism. By contrast, adipose research within other, closely related, teleost species has a rich and extensive history, owing to the economic importance of these fish as a food source. Here, we compare and contrast knowledge on peroxisome proliferator-activated receptor gamma (PPARG)-mediated adipogenesis derived from both biomedical and aquaculture literatures. We first concentrate on the biomedical literature to (i) briefly review PPARG-mediated adipogenesis in mammals, before (ii) reviewing Pparg-mediated adipogenesis in zebrafish. Finally, we (iii) mine the aquaculture literature to compare and contrast Pparg-mediated adipogenesis in aquaculturally relevant teleosts. Our goal is to highlight evolutionary similarities and differences in adipose biology that will inform our understanding of the role of adipose tissue in obesity and related disease.

Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling.

The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations.

The Lhx9-integrin pathway is essential for positioning of the proepicardial organ.

The development of the vertebrate embryonic heart occurs by hyperplastic growth as well as the incorporation of cells from tissues outside of the initial heart field. Amongst these tissues is the epicardium, a cell structure that develops from the precursor proepicardial organ on the right side of the septum transversum caudal to the developing heart. During embryogenesis, cells of the proepicardial organ migrate, adhere and envelop the maturing heart, forming the epicardium. The cells of the epicardium then delaminate and incorporate into the heart giving rise to cardiac derivatives, including smooth muscle cells and cardiac fibroblasts. Here, we demonstrate that the LIM homeodomain protein Lhx9 is transiently expressed in Xenopus proepicardial cells and is essential for the position of the proepicardial organ on the septum transversum. Utilizing a small-molecule screen, we found that Lhx9 acts upstream of integrin-paxillin signaling and consistently demonstrate that either loss of Lhx9 or disruption of the integrin-paxillin pathway results in mis-positioning of the proepicardial organ and aberrant deposition of extracellular matrix proteins. This leads to a failure of proepicardial cell migration and adhesion to the heart, and eventual death of the embryo. Collectively, these studies establish a requirement for the Lhx9-integrin-paxillin pathway in proepicardial organ positioning and epicardial formation.

Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure and Caenorhabditis elegans Gastrulation.

Neural tube defects including spina bifida are common and severe congenital disorders. In mice, mutations in more than 200 genes can result in neural tube defects. We hypothesized that this large gene set might include genes whose homologs contribute to morphogenesis in diverse animals. To test this hypothesis, we screened a set of Caenorhabditis elegans homologs for roles in gastrulation, a topologically similar process to vertebrate neural tube closure. Both C. elegans gastrulation and vertebrate neural tube closure involve the internalization of surface cells, requiring tissue-specific gene regulation, actomyosin-driven apical constriction, and establishment and maintenance of adhesions between specific cells. Our screen identified several neural tube defect gene homologs that are required for gastrulation in C. elegans, including the transcription factor sptf-3. Disruption of sptf-3 in C. elegans reduced the expression of early endodermally expressed genes as well as genes expressed in other early cell lineages, establishing sptf-3 as a key contributor to multiple well-studied C. elegans cell fate specification pathways. We also identified members of the actin regulatory WAVE complex (wve-1, gex-2, gex-3, abi-1, and nuo-3a). Disruption of WAVE complex members reduced the narrowing of endodermal cells' apical surfaces. Although WAVE complex members are expressed broadly in C. elegans, we found that expression of a vertebrate WAVE complex member, nckap1, is enriched in the developing neural tube of Xenopus. We show that nckap1 contributes to neural tube closure in Xenopus. This work identifies in vivo roles for homologs of mammalian neural tube defect genes in two manipulable genetic model systems.

A distinct mechanism of vascular lumen formation in Xenopus requires EGFL7.

During vertebrate blood vessel development, lumen formation is the critical process by which cords of endothelial cells transition into functional tubular vessels. Here, we use Xenopus embryos to explore the cellular and molecular mechanisms underlying lumen formation of the dorsal aorta and the posterior cardinal veins, the primary major vessels that arise via vasculogenesis within the first 48 hours of life. We demonstrate that endothelial cells are initially found in close association with one another through the formation of tight junctions expressing ZO-1. The emergence of vascular lumens is characterized by elongation of endothelial cell shape, reorganization of junctions away from the cord center to the periphery of the vessel, and onset of Claudin-5 expression within tight junctions. Furthermore, unlike most vertebrate vessels that exhibit specialized apical and basal domains, we show that early Xenopus vessels are not polarized. Moreover, we demonstrate that in embryos depleted of the extracellular matrix factor Epidermal Growth Factor-Like Domain 7 (EGFL7), an evolutionarily conserved factor associated with vertebrate vessel development, vascular lumens fail to form. While Claudin-5 localizes to endothelial tight junctions of EGFL7-depleted embryos in a timely manner, endothelial cells of the aorta and veins fail to undergo appropriate cell shape changes or clear junctions from the cell-cell contact. Taken together, we demonstrate for the first time the mechanisms by which lumens are generated within the major vessels in Xenopus and implicate EGFL7 in modulating cell shape and cell-cell junctions to drive proper lumen morphogenesis.

RNA-seq in the tetraploid Xenopus laevis enables genome-wide insight in a classic developmental biology model organism.

Advances in sequencing technology have significantly advanced the landscape of developmental biology research. The dissection of genetic networks in model and non-model organisms has been greatly enhanced with high-throughput sequencing technologies. RNA-seq has revolutionized the ability to perform developmental biology research in organisms without a published genome sequence. Here, we describe a protocol for developmental biologists to perform RNA-seq on dissected tissue or whole embryos. We start with the isolation of RNA and generation of sequencing libraries. We further show how to interpret and analyze the large amount of sequencing data that is generated in RNA-seq. We explore the abilities to examine differential expression, gene duplication, transcript assembly, alternative splicing and SNP discovery. For the purposes of this article, we use Xenopus laevis as the model organism to discuss uses of RNA-seq in an organism without a fully annotated genome sequence.

Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion.

Striated muscles that enable mouth opening and swallowing during feeding are essential for efficient energy acquisition, and are likely to have played a fundamental role in the success of early jawed vertebrates. The developmental origins and genetic requirements of these muscles are uncertain. Here, we determine by indelible lineage tracing in mouse that fibres of sternohyoid muscle (SHM), which is essential for mouth opening during feeding, and oesophageal striated muscle (OSM), which is crucial for voluntary swallowing, arise from Pax3-expressing somite cells. In vivo Kaede lineage tracing in zebrafish reveals the migratory route of cells from the anteriormost somites to OSM and SHM destinations. Expression of pax3b, a zebrafish duplicate of Pax3, is restricted to the hypaxial region of anterior somites that generate migratory muscle precursors (MMPs), suggesting that Pax3b plays a role in generating OSM and SHM. Indeed, loss of pax3b function led to defective MMP migration and OSM formation, disorganised SHM differentiation, and inefficient ingestion and swallowing of microspheres. Together, our data demonstrate Pax3-expressing somite cells as a source of OSM and SHM fibres, and highlight a conserved role of Pax3 genes in the genesis of these feeding muscles of vertebrates.

Tcf21 regulates the specification and maturation of proepicardial cells.

The epicardium is a mesothelial cell layer essential for vertebrate heart development and pertinent for cardiac repair post-injury in the adult. The epicardium initially forms from a dynamic precursor structure, the proepicardial organ, from which cells migrate onto the heart surface. During the initial stage of epicardial development crucial epicardial-derived cell lineages are thought to be determined. Here, we define an essential requirement for transcription factor Tcf21 during early stages of epicardial development in Xenopus, and show that depletion of Tcf21 results in a disruption in proepicardial cell specification and failure to form a mature epithelial epicardium. Using a mass spectrometry-based approach we defined Tcf21 interactions and established its association with proteins that function as transcriptional co-repressors. Furthermore, using an in vivo systems-based approach, we identified a panel of previously unreported proepicardial precursor genes that are persistently expressed in the epicardial layer upon Tcf21 depletion, thereby confirming a primary role for Tcf21 in the correct determination of the proepicardial lineage. Collectively, these studies lead us to propose that Tcf21 functions as a transcriptional repressor to regulate proepicardial cell specification and the correct formation of a mature epithelial epicardium.

ROCKs cause SHP-wrecks and broken hearts.

During embryogenesis, the heart is one of the first organs to develop. Its formation requires a complex combination of migration of cardiac precursors to the ventral midline coupled with the fusion of these cardiogenic fields and subsequent cellular reorganization to form a linear heart tube. A finely controlled choreography of cell proliferation, adhesion, contraction and movement drives the heart tube to loop and subsequently septate to form the four-chambered mammalian heart we are familiar with. Defining how this plethora of cellular processes is controlled both spatially and temporally is a scientific feat that has fascinated researchers for decades. Unfortunately, the complex nature of this organ's development also makes it a prime target for mutation-induced malformation, as evidenced by the multitude of prevalent congenital heart disorders identified that afflict up to 1% of the population.

Morpholino injection in Xenopus.

The study of gene function in developmental biology has been significantly furthered by advances in antisense technology made in the early 2000s. This was achieved, in particular, by the introduction of morpholino (MO) oligonucleotides. The introduction of antisense MO oligonucleotides into cells enables researchers to readily reduce the levels of their protein of interest without investing huge financial or temporal resources, in both in vivo and in vitro model systems. Historically, the African clawed frog Xenopus has been used to study vertebrate embryological development, due to its ability to produce vast numbers of offspring that develop rapidly, in synchrony, and can be cultured in buffers with ease. The developmental progress of Xenopus embryos has been extensively characterized and this model organism is very easy to maintain. It is these attributes that enable MO-based knockdown strategies to be so effective in Xenopus. In this chapter, we will detail the methods of microinjecting MO oligonucleotides into early embryos of X. laevis and X. tropicalis. We will discuss how MOs can be used to prevent either pre-mRNA splicing or translation of the specific gene of interest resulting in abrogation of that gene's function and advise on what control experiments should be undertaken to verify their efficacy.

SHP-2 acts via ROCK to regulate the cardiac actin cytoskeleton.

Noonan syndrome is one of the most common causes of human congenital heart disease and is frequently associated with missense mutations in the protein phosphatase SHP-2. Interestingly, patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), juvenile myelomonocytic leukemia (JMML) and LEOPARD syndrome frequently carry a second, somatically introduced subset of missense mutations in SHP-2. To determine the cellular and molecular mechanisms by which SHP-2 regulates heart development and, thus, understand how Noonan-associated mutations affect cardiogenesis, we introduced SHP-2 encoding the most prevalent Noonan syndrome and JMML mutations into Xenopus embryos. Resulting embryos show a direct relationship between a Noonan SHP-2 mutation and its ability to cause cardiac defects in Xenopus; embryos expressing Noonan SHP-2 mutations exhibit morphologically abnormal hearts, whereas those expressing an SHP-2 JMML-associated mutation do not. Our studies indicate that the cardiac defects associated with the introduction of the Noonan-associated SHP-2 mutations are coupled with a delay or arrest of the cardiac cell cycle in M-phase and a failure of cardiomyocyte progenitors to incorporate into the developing heart. We show that these defects are a result of an underlying malformation in the formation and polarity of cardiac actin fibers and F-actin deposition. We show that these defects can be rescued in culture and in embryos through the inhibition of the Rho-associated, coiled-coil-containing protein kinase 1 (ROCK), thus demonstrating a direct relationship between SHP-2(N308D) and ROCK activation in the developing heart.

Xenopus: An emerging model for studying congenital heart disease.

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes.

The emergence of ectomesenchyme.

In the head, neural crest cells generate ectomesenchymal derivatives: cartilage, bone, and connective tissue. Indeed, these cells generate much of the cranial skeleton. There have, however, been few studies of how this lineage is established. Here, we show that neural crest cells stop expressing early neural crest markers upon entering the pharyngeal arches and switch to become ectomesenchymal. By contrast, those neural crest cells that do not enter the arches persist in their expression of early neural crest markers. We further show that fibroblast growth factor (FGF) signaling is involved in directing neural crest cells to become ectomesenchymal. If neural crest cells are rendered insensitive to FGFs, they persist in their expression of early neural crest markers, even after entering the pharyngeal arches. However, our results further suggest that, although FGF signaling is required for the realization of the ectomesenchymal lineages, other cues from the pharyngeal epithelia are also likely to be involved.

The differential regulation of Lck kinase phosphorylation sites by CD45 is critical for T cell receptor signaling responses.

The molecular mechanisms whereby the CD45 tyrosine phosphatase (PTPase) regulates T cell receptor (TCR) signaling responses remain to be elucidated. To investigate this question, we have reconstituted CD45 (encoded by Ptprc)-deficient mice, which display severe defects in thymic development, with five different expression levels of transgenic CD45RO, or with mutant PTPase null or PTPase-low CD45R0. Whereas CD45 PTPase activity was absolutely required for the reconstitution of thymic development, only 3% of wild-type CD45 activity restored T cell numbers and normal cytotoxic T cell responses. Lowering the CD45 expression increased CD4 lineage commitment. Peripheral T cells with very low activity of CD45 phosphatase displayed reduced TCR signaling, whereas intermediate activity caused hyperactivation of CD4+ and CD8+ T cells. These results are explained by a rheostat mechanism whereby CD45 differentially regulates the negatively acting pTyr-505 and positively acting pTyr-394 p56(lck) tyrosine kinase phosphorylation sites. We propose that high wild-type CD45 expression is necessary to dephosphorylate p56(lck) pTyr-394, suppressing CD4 T+ cell lineage commitment and hyperactivity.

Lateral response dynamics and hemispheric dominance for speech perception.

In this study, we investigated the mechanism for the left cerebral hemisphere's dominance for speech perception. We utilized the crossover of auditory pathways in the central nervous system to present speech stimuli more directly to the left hemisphere (via the right ear) and right hemisphere (via the left ear). Using functional MRI, we found that estimated duration of neural response in the left auditory cortex increased as more speech information was directly received from the right ear. Conversely, response duration in the right auditory cortex was not modulated when more speech information was directly received from the left ear. These data suggest that selective temporal responding distinguishes the dominant from nondominant hemisphere of the human brain during speech perception.