Strategies for analyzing cardiac phenotypes in the zebrafish embryo.

The molecular mechanisms underlying cardiogenesis are of critical biomedical importance due to the high prevalence of cardiac birth defects. Over the past two decades, the zebrafish has served as a powerful model organism for investigating heart development, facilitated by its powerful combination of optical access to the embryonic heart and plentiful opportunities for genetic analysis. Work in zebrafish has identified numerous factors that are required for various aspects of heart formation, including the specification and differentiation of cardiac progenitor cells, the morphogenesis of the heart tube, cardiac chambers, and atrioventricular canal, and the establishment of proper cardiac function. However, our current roster of regulators of cardiogenesis is by no means complete. It is therefore valuable for ongoing studies to continue pursuit of additional genes and pathways that control the size, shape, and function of the zebrafish heart. An extensive arsenal of techniques is available to distinguish whether particular mutations, morpholinos, or small molecules disrupt specific processes during heart development. In this chapter, we provide a guide to the experimental strategies that are especially effective for the characterization of cardiac phenotypes in the zebrafish embryo.

Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition mechanism.

Retinoic acid (RA) is an important developmental signaling molecule responsible for the patterning of multiple vertebrate tissues. RA is also a potent teratogen, causing multi-organ birth defects in humans. Endogenous RA levels must therefore be tightly controlled in the developing embryo. We used a microarray approach to identify genes that function as negative feedback regulators of retinoic acid signaling. We screened for genes expressed in early somite-stage embryos that respond oppositely to treatment with RA versus RA antagonists and validated them by RNA in situ hybridization. Focusing on genes known to be involved in RA metabolism, we determined that dhrs3a, which encodes a member of the short-chain dehydrogenase/reductase protein family, is both RA dependent and strongly RA inducible. Dhrs3a is known to catalyze the reduction of the RA precursor all-trans retinaldehyde to vitamin A; however, a developmental function has not been demonstrated. Using morpholino knockdown and mRNA over-expression, we demonstrate that Dhrs3a is required to limit RA levels in the embryo, primarily within the central nervous system. Dhrs3a is thus an RA-induced feedback inhibitor of RA biosynthesis. We conclude that retinaldehyde availability is an important level at which RA biosynthesis is regulated in vertebrate embryos.

Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts.

Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.

Cardiac patterning and morphogenesis in zebrafish.

Development of the embryonic vertebrate heart requires the precise coordination of pattern formation and cell movement. Taking advantage of the availability of zebrafish mutations that disrupt cardiogenesis, several groups have identified key regulators of specific aspects of cardiac patterning and morphogenesis. Several genes, including gata5, fgf8, bmp2b, one-eyed pinhead, and hand2, have been shown to be relevant to the patterning events that regulate myocardial differentiation. Studies of mutants with morphogenetic defects have indicated at least six genes that are essential for cardiac fusion and heart tube assembly, including casanova, bonnie and clyde, gata5, one-eyed pinhead, hand2, miles apart, and heart and soul. Furthermore, analysis of the jekyll gene has indicated its important role during the morphogenesis of the atrioventricular valve. Altogether, these data provide a substantial foundation for future investigations of cardiac patterning, cardiac morphogenesis, and the relationship between these processes.

casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish.

Early endoderm formation in zebrafish requires at least three loci that function downstream of Nodal signaling but upstream of the early endodermal marker sox17: bonnie and clyde (bon), faust (fau), and casanova (cas). cas mutants show the most severe phenotype as they do not form any gut tissue and lack all sox17 expression. Activation of the Nodal signaling pathway or overexpression of Bon or Fau/Gata5 fails to restore any sox17 expression in cas mutants, demonstrating that cas plays a central role in endoderm formation. Here we show that cas encodes a novel member of the Sox family of transcription factors. Initial cas expression appears in the dorsal yolk syncytial layer (YSL) in the early blastula, and is independent of Nodal signaling. In contrast, endodermal expression of cas, which begins in the late blastula, is regulated by Nodal signaling. Cas is a potent inducer of sox17 expression in wild-type embryos as well as in bon and fau/gata5 mutants. Cas is also a potent inducer of sox17 expression in MZoep mutants, which cannot respond to Nodal signaling. In addition, ectopic expression of cas in presumptive mesodermal cells leads to their transfating into endoderm. Altogether, these data indicate that Cas is the principal transcriptional effector of Nodal signaling during zebrafish endoderm formation.

The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development.

The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. Here, we demonstrate that the zebrafish hands off locus is essential for the development of two structures derived from the lateral plate mesoderm - the heart and the pectoral fin. hands off mutant embryos have defects in myocardial development from an early stage: they produce a reduced number of myocardial precursors, and the myocardial tissue that does form is improperly patterned and fails to maintain tbx5 expression. A similar array of defects is observed in the differentiation of the pectoral fin mesenchyme: small fin buds form in a delayed fashion, anteroposterior patterning of the fin mesenchyme is absent and tbx5 expression is poorly maintained. Defects in these mesodermal structures are preceded by the aberrant morphogenesis of both the cardiogenic and forelimb-forming regions of the lateral plate mesoderm. Molecular analysis of two hands off alleles indicates that the hands off locus encodes the bHLH transcription factor Hand2, which is expressed in the lateral plate mesoderm starting at the completion of gastrulation. Thus, these studies reveal early functions for Hand2 in several cellular processes and highlight a genetic parallel between heart and forelimb development.

The zebrafish bonnie and clyde gene encodes a Mix family homeodomain protein that regulates the generation of endodermal precursors.

Vertebrate endoderm development has recently become the focus of intense investigation. In this report, we first show that the zebrafish bonnie and clyde (bon) gene plays a critical early role in endoderm formation. bon mutants exhibit a profound reduction in the number of sox17-expressing endodermal precursors formed during gastrulation, and, consequently, a profound reduction in gut tissue at later stages. The endodermal precursors that do form in bon mutants, however, appear to differentiate normally indicating that bon is not required at later steps of endoderm development. We further demonstrate that bon encodes a paired-class homeodomain protein of the Mix family that is expressed transiently before and during early gastrulation in both mesodermal and endodermal progenitors. Overexpression of bon can rescue endodermal gene expression and the formation of a gut tube in bon mutants. Analysis of a newly identified mutant allele reveals that a single amino acid substitution in the DNA recognition helix of the homeodomain creates a dominant interfering form of Bon when overexpressed. We also show through loss- and gain-of-function analyses that Bon functions exclusively downstream of cyclops and squint signaling. Together, our data demonstrate that Bon is a critical transcriptional regulator of early endoderm formation.

A conserved role for H15-related T-box transcription factors in zebrafish and Drosophila heart formation.

T-box transcription factors are critical regulators of early embryonic development. We have characterized a novel zebrafish T-box transcription factor, hrT (H15-related T box) that is a close relative of Drosophila H15 and a recently identified human gene. We show that Drosophila H15 and zebrafish hrT are both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrT expression in anterior lateral plate mesoderm suggest a very early role for hrT in the differentiation of the cardiac precursors. hrT is coexpressed with gata4 and nkx2.5 not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, we analyzed hrT expression in pandora mutant embryos, since these have defects in many of the tissues that express hrT, including the heart. hrT expression is much reduced in the early heart fields of pandora mutants, whereas it is ectopically expressed subsequently. Using hrT expression as a marker, we describe a midline patterning defect in pandora affecting the anterior hindbrain and associated midline mesendodermal derivatives. We discuss the possibility that the cardiac ventricular defect previously described in pandora and the midline defects described here are related.

Gata5 is required for the development of the heart and endoderm in zebrafish.

The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Here we present evidence from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. We demonstrate that zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5.

Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.

The embryonic vertebrate heart is divided into two major chambers, an anterior ventricle and a posterior atrium. Although the fundamental differences between ventricular and atrial tissues are well documented, it is not known when and how cardiac anterior-posterior (A-P) patterning occurs. The expression patterns of two zebrafish cardiac myosin genes, cardiac myosin light chain 2 (cmlc2) and ventricular myosin heavy chain (vmhc), allow us to distinguish two populations of myocardial precursors at an early stage, well before the heart tube forms. These myocardial subpopulations, which may represent the ventricular and atrial precursors, are organized in a medial-lateral pattern within the precardiac mesoderm. Our examinations of cmlc2 and vmhc expression throughout the process of heart tube assembly indicate the important role of an intermediate structure, the cardiac cone, in the conversion of this early medial-lateral pattern into the A-P pattern of the heart tube. To gain insight into the genetic regulation of heart tube assembly and patterning, we examine cmlc2 and vmhc expression in several zebrafish mutants. Analyses of mutations that cause cardia bifida demonstrate that the achievement of a proper cardiac A-P pattern does not depend upon cardiac fusion. On the other hand, cardiac fusion does not ensure the proper A-P orientation of the ventricle and atrium, as demonstrated by the heart and soul mutation, which blocks cardiac cone morphogenesis. Finally, the pandora mutation interferes with the establishment of the early medial-lateral myocardial pattern. Altogether, these data suggest new models for the mechanisms that regulate the formation of a patterned heart tube and provide an important framework for future analyses of zebrafish mutants with defects in this process.

Patterning during organogenesis: genetic analysis of cardiac chamber formation.

A classical genetic approach, in which mutagenized organisms are screened for phenotypes of interest, is appealing for the analysis of developmental processes. Here, we describe the advantages of zebrafish genetics for the study of heart development. As an example of the utility of this strategy, we discuss its potential to illuminate the molecular mechanisms of cardiac chamber formation. The signals that specify ventricular and atrial lineages and the differentiation pathways that produce distinct chambers are poorly understood. Recently identified zebrafish mutations that disrupt ventricular or atrial development promise to reveal genes essential for these processes.

Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development.

The T cell coreceptors CD4 and CD8 enhance T cell responses to TCR signals by participating in complexes containing TCR, coreceptor, and MHC molecules. These ternary complexes are also hypothesized to play a seminal role during T cell development, although the precise timing, frequency, and consequences of TCR-coreceptor-MHC interactions during positive selection and lineage commitment remain unclear. To address these issues, we designed transgenic mice expressing mutant I-Ek molecules with reduced CD4-binding capability. These transgenic lines were crossed to three different lines of I-Ek-specific TCR transgenic mice, and the efficiency of production of CD4+ lineage cells in the doubly transgenic progeny was assessed. Surprisingly, replacing wild-type I-Ek molecules with these mutant molecules did not affect the production of CD4+CD8- thymocytes or CD4+ peripheral T cells expressing any of the three TCRs examined. These data, when considered together with other experiments addressing the role of coreceptor during development, suggest that not all MHC class II-specific thymocytes require optimal and simultaneous TCR-CD4-MHC interactions to mature. Alternatively, it is possible that these particular alterations of I-Ek do not disrupt the CD4-MHC interaction adequately, potentially indicating functional differences between I-A and I-E MHC class II molecules.

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning.

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart.

Structurally similar TCRs differ in their efficiency of positive selection.

Studies of TCR transgenic mice have demonstrated that in these systems positive selection is not an efficient process. The capacity of the thymus to produce mature T cells is limited, even when all immature thymocytes express appropriate Ag receptors. Analysis of TCR transgenic mice expressing reduced levels of MHC molecules have shown that MHC surface density can be a limiting factor during development. Whether peptide availability in the thymus also limits the efficiency of positive selection remains controversial. Here, we examine the efficiency of positive selection in three similar lines of TCR transgenic mice, all of which express V alpha11/beta3+ TCRs specific for cytochrome c peptides bound to I-Ek. We demonstrate that thymocytes expressing these similar TCRs mature with very different efficiencies in H-2k mice. Furthermore, efficient positive selection of thymocytes expressing these three TCRs varies in its dependence on MHC density. These data suggest that similar TCRs can differ in their degree of specificity for peptide/MHC complexes during positive selection; some TCRs may require specific complexes for selection while other TCRs are more promiscuous in their thymic interactions. Alternatively, these three TCRs may differ in their affinities for positively selecting ligands.

In vivo functions mediated by the p41 isoform of the MHC class II-associated invariant chain.

We used a "hit and run" gene targeting strategy to generate mice expressing only the p41 isoform of the conserved invariant (Ii) chain associated with MHC class II molecules. In contrast to mutants expressing only p31 Ii chain, a small proportion of A(alpha)b A(beta)b molecules produced by these animals have reduced mobilities in SDS-PAGE and appear incompletely processed. Nonetheless, class II surface expression, peptide occupancy, CD4+ T cell maturation, and proliferative responses toward intact protein Ags are efficiently reconstituted. Moreover, spleen cells exclusively expressing p41 or p31 alone display equivalent dose-response curves in Ag presentation assays. Similar conclusions were reached analyzing mutants expressing two independent MHC haplotypes. Overall, these results demonstrate that Ii chain functional activities as a class II-specific chaperone are largely shared by p31 and p41 isoforms in the intact animal. Mutant mouse strains producing only p31 or p41 under control of endogenous regulatory elements responsible for constitutive and inducible Ii chain expression should prove useful for dissecting the contributions of these isoforms to diverse CD4+ T cell responses in vivo, such as those responsible for Ab production, inflammatory responses, autoimmune diseases, and protection against infectious agents.

Alterations in CD4 dependence accompany T cell development and differentiation.

Several studies have indicated that the necessity for co-receptor engagement during T cell activation depends on the avidity of the TCR-MHC interaction under investigation. Using thymocytes, naive T cells and a long-term T cell line isolated from 2B4 TCR transgenic mice, we have examined the role of the CD4 co-receptor on cells expressing the identical TCR at multiple stages of T cell maturation. When anti-CD4 Fab fragments were used to block CD4-MHC class II interactions, we found decreasing CD4 dependence as T cells matured. As a second approach to examining the role of the CD4 co-receptor, we generated I-Ek mutants defective in CD4 interactions. In the course of this study, we identified a new potential site for CD4 interaction in the beta1 domain of I-Ek. The new beta1 mutation and a mutation in the previously described CD4 binding site in the beta2 domain both interfere with stimulation of 2B4 thymocytes, but not mature T cells. Together these data demonstrate that the role of the CD4 co-receptor depends on the state of maturation of the T cell.

Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42.

The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

Do the CD4 and CD8 lineages represent parallel pathways?

It is now well established that the progression of T cell development requires interactions between the surfaces of thymocytes and thymic stromal cells. For example, the maturation of CD4+8+ cells into functional CD4+ or CD8+ cells requires TCR/MHC interactions, which, depending on the specificity of the particular TCR, direct the thymocyte to the appropriate lineage. Beyond this, little is known about the molecular mechanism of this lineage choice. Here we describe our recent studies of CD4/CD8 lineage commitment using TCR transgenic mice expressing a well-defined MHC class II specific TCR. While the results of these experiments are inconsistent with a model in which CD4 versus CD8 lineage is determined by an initial TCR/MHC/co-receptor interaction, they also do not support a simple stochastic model of lineage commitment. Instead, we suggest that the CD4 and CD8 lineage may not represent equivalent pathways of T cell maturation. Additionally, we draw several parallels between stochastic models in positive selection and in hematopoiesis.

Phylogeny of the Bovidae (Artiodactyla, Mammalia), based on mitochondrial ribosomal DNA sequences.

Portions of the 12S and 16S mitochondrial ribosomal genes for 16 species representing nine tribes in the mammal family Bovidae were compared with six previously published orthologous sequences. Phylogenetic analysis of variable nucleotide positions under different constraints and weighting schemes revealed no robust groupings among tribes. Consensus trees support previous hypotheses of monophyly for four clades, including the traditional subfamily Bovinae. However, the basal diversification of bovid tribes, which was largely unresolved by morphological, immunodiffusion, allozyme, and protein sequence data, remains unresolved with the addition of DNA sequence data. The intractability of this systematic problem is consistent with a rapid radiation of the major bovid groups. Several analyses of our data show that monophyly of the Bovidae, which was weakly supported by previous morphological and molecular work, is questionable.