New Insights Into the Role of RNA-Binding Proteins in the Regulation of Heart Development.

The regulation of gene expression during development takes place both at the transcriptional and posttranscriptional levels. RNA-binding proteins (RBPs) regulate pre-mRNA processing, mRNA localization, stability, and translation. Many RBPs are expressed in the heart and have been implicated in heart development, function, or disease. This chapter will review the current knowledge about RBPs in the developing heart, focusing on those that regulate posttranscriptional gene expression. The involvement of RBPs at each stage of heart development will be considered in turn, including the establishment of specific cardiac cell types and formation of the primitive heart tube, cardiac morphogenesis, and postnatal maturation and aging. The contributions of RBPs to cardiac birth defects and heart disease will also be considered in these contexts. Finally, the interplay between RBPs and other regulatory factors in the developing heart, such as transcription factors and miRNAs, will be discussed.

Diversity and conservation of CELF1 and CELF2 RNA and protein expression patterns during embryonic development.

INTRODUCTION: CUG-BP, Elav-like family member 1 (CELF1) and CELF2 are RNA-binding proteins that regulate several stages of RNA processing, and are broadly expressed in developing and adult tissues. In this study, we investigated the expression patterns of CELF1 and CELF2 transcripts and proteins in different tissues, stages of development, and organisms. RESULTS: We found that CELF1 and CELF2 protein levels are regulated independently of transcript levels during heart development, and these proteins exhibit nuclear and cytoplasmic isoforms in the embryonic heart. We found that the subcellular distribution of CELF1 differs between heart, liver, nervous system, and eye, and identified tissue-specific isoforms of both CELF1 and CELF2 in these tissues. CELF1 and CELF2 are largely co-expressed, but are found in mutually exclusive territories in several organs, including the heart and eye. Finally, we show that the expression patterns observed in embryonic chicken were mostly recapitulated in the developing mouse, suggesting that the roles of these proteins in the tissues and cells of the developing embryo are conserved as well. CONCLUSIONS: CELF1 and CELF2 may underlie conserved, developmentally regulated, tissue-specific processes in vertebrate embryos. Different tissues likely have unique profiles of nuclear and cytoplasmic CELF1- and CELF2-mediated functions.

The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing.

Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.

Regulation of avian cardiac myogenesis by activin/TGFbeta and bone morphogenetic proteins.

Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and mediated by activin or a related molecule and an endoderm-derived signal acting on mesoderm and involving BMP-2. In this study, experiments were designed to investigate the temporal relationship between these signaling events and the potential role of other TGFbeta superfamily members in regulating early steps of heart muscle development. We find that while activin or TGFbeta can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2 or BMP-4, in combination with FGF-4, can readily induce cardiac myocyte formation in posterior mesoderm, but shows no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFbeta and BMP-2/BMP-4 therefore have distinct and reciprocal cardiac-inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and anterior lateral endoderm, respectively. Experiments with noggin and follistatin provide additional evidence indicating that BMP signaling lies downstream of an activin/TGFbeta signal in the cardiac myogenesis pathway. In contrast to the cardiogenic-inducing capacities of BMP-2/BMP-4 in mesoderm, however, we find that BMP-2 or BMP-4 inhibits cardiac myogenesis prior to stage 3, demonstrating multiple roles for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFbeta signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, and mediated in part by BMPs, act on emerging mesoderm within cardiogenic regions to activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation.

Effects of epidermal growth factor (EGF) and prolactin on EGF receptor cytoskeletal association in mammary epithelial cells.

Prolactin treatment of NMuMG mammary epithelial cells inhibits the ability of epidermal growth factor (EGF) to transduce a variety of signals, possibly by interfering with receptor tyrosine phosphorylation. However, the mechanism by which prolactin inhibits EGF receptor signaling is unclear. The objective of this study was to evaluate the effects of prolactin on the dynamics of EGF receptor degradation and resynthesis, and on the association of the receptor with the cytoskeleton. EGF decreased the EGF receptor content of NMuMG cells, and this decrease was unaffected by prolactin treatment. Subsequent to the decrease in EGF receptors, cells reaccumulated EGF receptors, and this re-accumulation was also unaffected by prolactin. In other studies, EGF induced a rapid association of EGF receptor with Triton X-100-insoluble (cytoskeletal) elements. The cytoskeletally associated receptors were more heavily tyrosine phosphorylated than soluble receptors in the absence of prolactin. In the presence of prolactin, similar amounts of EGF receptor associated with the cytoskeleton, but both cytoskeletal and soluble receptors exhibited decreased tyrosine phosphorylation. These studies indicate that the effects of prolactin on EGF receptor signaling are not likely to be due to altered receptor dynamics or cytoskeletal association but are more likely due to an alteration in receptor kinase activity.

Induction of cardiac myogenesis in avian pregastrula epiblast: the role of the hypoblast and activin.

An in vitro assay has been developed to investigate tissue interactions regulating myocardial cell specification in birds. Explants from the posterior region of stage XI-XIV blastulas were found to form heart muscle at high frequency with a timing that corresponded to onset of cardiac myocyte differentiation in vivo. Isolation and recombination experiments demonstrated that a signal from the hypoblast was required to induce cardiac myogenesis in the epiblast, and regional differences in epiblast responsiveness and hypoblast inductiveness restrict appearance of cardiac myocytes to the posterior region. Explantation studies provided evidence that myocardial cell specification is underway by stage 3, indicating that the hypoblast-derived signal occurs shortly before specification is detected. Recombinations were also performed to compare cardiac-inducing capacities of pregastrula hypoblast and stage 5 anterior lateral endoderm. The hypoblast possessed broad capacity to induce heart muscle cells in pregastrula and mid-gastrula epiblast, and modest ability to induce cardiac myogenesis in stage 4 posterior primitive streak. Stage 5 anterior lateral endoderm, in contrast, showed no ability to induce heart development in epiblast cells but was a potent inducer of cardiac myogenesis in cells from stage 4 posterior primitive streak. These findings suggest that the hypoblast-derived signal likely acts upstream of proposed heart-inducing signals provided by anterior lateral endoderm. Experiments were also performed to investigate whether activin, or an activin-like molecule, is involved in regulating cardiac myogenesis. Follistatin blocked cardiac myogenesis in stage XI-XIV posterior region explants and activin induced cardiac myogenesis in a dose-dependent fashion in posterior epiblast. These findings indicate that activin, or an activin-like molecule, is required for and is sufficient to stimulate cardiac myogenesis in posterior region pregastrula epiblast. Three models are presented to explain these results.

The ethylene response mediator ETR1 from Arabidopsis forms a disulfide-linked dimer.

Mutations in the ETR1 gene of the higher plant Arabidopsis confer insensitivity to ethylene, indicating a role for the gene product in ethylene signal perception and transduction. The ETR1 gene product has an amino-terminal hydrophobic domain and a carboxyl-terminal domain showing homology to the two-component signal transduction proteins of bacteria. We report here that in both its native Arabidopsis and when transgenically expressed in yeast, the ETR1 protein is isolated from membranes as a dimer of 147 kDa. Treatment with the reducing agent dithiothreitol converted the dimer to a monomer of 79 kDa, indicative of a disulfide linkage between monomers. Expression of truncated versions of ETR1 in yeast confirmed that the high molecular mass form is a homodimer and demonstrated that the amino-terminal region of ETR1 is necessary and sufficient for this dimerization. Site-directed mutagenesis of two cysteines near the amino terminus of ETR1 prevented formation of the covalently linked dimer in yeast, consistent with a role in disulfide bond formation. These data indicate that ETR1 may use a dimeric mechanism of signal transduction in a manner similar to its bacterial counterparts but with the additional feature of a disulfide bond between monomers.