Norma Towers

MEF‐2 function is modified by a novel co‐repressor, MITR

The MEF‐2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF‐2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage‐specific cofactors may also underlie MEF‐2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two‐hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF‐2 interacting transcription repressor (MITR) protein binds to the N‐terminal MADS/MEF‐2 region of the MEF‐2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF‐2‐dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co‐repressor, recruiting a specific deacetylase to downregulate MEF‐2 activity.

Muscle-specific expression of SRF-related genes in the early embryo of Xenopus laevis

We have isolated two members of the RSRF protein family, SL‐1 and SL‐2, in Xenopus laevis. Both proteins contain SRF‐type DNA binding domains and are related to the human protein, RSRFC4. SL‐1 constitutes a novel member of the RSRF family whilst SL‐2 is similar to human RSRFC4 throughout its length. SL‐1 protein recognizes the consensus DNA sequence CTA(A/T)4TAR in vitro and can bind to the same regulatory sites as other A/T‐rich sequence‐specific binding activities, such as the muscle‐specific regulatory factor, MEF‐2. Transcription of both Xenopus genes is restricted to the somitic mesoderm of early embryos and subsequently to the body muscle (myotomes) of the tadpole. In contrast, both genes are expressed ubiquitously in the adult frog. A binding activity, antigenically related to both human RSRFC4 and the SL‐2 gene product, is detected in Xenopus embryos and after gastrulation is localized to embryonic muscle. An indistinguishable binding activity is detected in many adult frog tissues. We conclude that the RSRF genes undergo a dramatic switch in their patterns of expression during development. We suggest that RSRF proteins may regulate muscle‐specific transcription in embryos, but acquire other roles during the course of development.

XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus

Phagocytic myeloid cells provide the principle line of immune defence during early embryogenesis in lower vertebrates. They may also have important functions during normal embryo morphogenesis, not least through the phagocytic clearance of cell corpses arising from apoptosis. We have identified two cDNAs that provide sensitive molecular markers of embryonic leukocytes in the early Xenopus embryo. These encode a peroxidase (XPOX2) and a Ly-6/uPAR-related protein (XLURP-1). We show that myeloid progenitors can first be detected at an antero-ventral site in early tailbud stage embryos (a region previously termed the anterior ventral blood island) and transiently express the haematopoetic transcription factors SCL and AML. Phagocytes migrate from this site along consistent routes and proliferate, becoming widely distributed throughout the tadpole long before the circulatory system is established. This migration can be followed in living embryos using a 5 kb portion of the XLURP-1 promoter to drive expression of EGFP specifically in the myeloid cells. Interestingly, whilst much of this migration occurs by movement of individual cells between embryonic germ layers, the rostral-most myeloid cells apparently migrate in an anterior direction along the ventral midline within the mesodermal layer itself. The transient presence of such cells as a strip bisecting the cardiac mesoderm immediately prior to heart tube formation suggests that embryonic myeloid cells may play a role in early cardiac morphogenesis.

Transcriptional regulation of the cardiac-specific MLC2 gene during Xenopus embryonic development.

The mechanisms by which transcription factors, which are not themselves tissue restricted, establish cardiomyocyte-specific patterns of transcription in vivo are unknown. Nor do we understand how positional cues are integrated to provide regionally distinct domains of gene expression within the developing heart. We describe regulation of the Xenopus XMLC2 gene, which encodes a regulatory myosin light chain of the contractile apparatus in cardiac muscle. This gene is expressed from the onset of cardiac differentiation in the frog embryo and is expressed throughout all the myocardium, both before and after heart chamber formation. Using transgenesis in frog embryos, we have identified an 82 bp enhancer within the proximal promoter region of the gene that is necessary and sufficient for heart-specific expression of an XMLC2 transgene. This enhancer is composed of two GATA sites and a composite YY1/CArG-like site. We show that the low-affinity SRF site is essential for transgene expression and that cardiac-specific expression also requires the presence of at least one adjacent GATA site. The overlapping YY1 site within the enhancer appears to act primarily as a repressor of ectopic expression, although it may also have a positive role. Finally, we show that the frog MLC2 promoter drives pan myocardial expression of a transgene in mice, despite the more restricted patterns of expression of murine MLC2 genes. We speculate that a common regulatory mechanism may be responsible for pan-myocardial expression of XMLC2 in both the frog and mouse, modulation of which could have given rise to more restricted patterns of expression within the heart of higher vertebrates.

Xenopus eHAND: a marker for the developing cardiovascular system of the embryo that is regulated by bone morphogenetic proteins

The bHLH protein eHAND is a sensitive marker for cardiovascular precursors in the Xenopus embryo. The earliest site of expression is a broad domain within the lateral plate mesoderm of the tailbud embryo. This domain comprises precursors that contribute to the posterior cardinal veins in later stages. Surprisingly, expression is profoundly asymmetric at this stage and is random with respect to embryo side. XeHAND is also expressed in an anterior domain that encompasses the prospective heart region. Within the myocardium and pericardium, transcripts are also asymmetrically distributed, but in these tissues they are localised in a left-sided manner. Later in development XeHAND transcripts are largely restricted to the ventral aorta, aortic arches and venous inflow tract (sinus venosus) which flank the heart itself, but no expression is detected in neural crest derivatives at any stage. This demonstrates that patterns of XeHAND expression differ markedly amongst vertebrates and that in Xenopus, XeHAND expression identifies all of the earliest formed elements of the cardiovascular system. In animal cap explants, expression of XeHAND (but not other markers of cardiogenic differentiation) is strongly induced by ectopic expression of the TGFbeta family members, BMP-2 and BMP-4, but this can be blocked by coexpression of a dominant negative BMP receptor. This suggests that XeHAND expression in the embryo is regulated by the ventralising signals of bone morphogenetic proteins. High levels of expression are also detected in explants treated with high doses of activin A which induces cardiac muscle differentiation. No such effect is seen with lower doses of activin, indicating that a second pathway may regulate the XeHAND gene during cardiogenesis.

The MLC1v gene provides a transgenic marker of myocardium formation within developing chambers of the Xenopus heart.

Many details of cardiac chamber morphogenesis could be revealed if muscle fiber development could be visualized directly within the hearts of living vertebrate embryos. To achieve this end, we have used the active promoter of the MLC1v gene to drive expression of green fluorescent protein (GFP) in the developing tadpole heart. By using a line of Xenopus laevis frogs transgenic for the MLC1v‐EGFP reporter, we have observed regionalized patterns of muscle formation within the ventricular chamber and maturation of the atrial chambers, from the onset of chamber formation through to the adult frog. In f1 generation MLC1v‐EGFP animals, promoter activity is first detected within the looping heart tube and delineates the forming ventricular chamber and proximal outflow tract throughout their development. The 8‐kb MLC1v promoter faithfully reproduces the embryonic expression of the endogenous MLC1v mRNA. At later larval stages, weak patches of EGFP fluorescence are found on the atrial side of the atrioventricular boundary. Subsequently, an extensive lattice of MLC1v‐expressing fibers extend across the mature atrial chambers of adult frog hearts and the transgene reveals the differing arrangement of muscle fibers in chamber versus outflow myocardium. The complete activity of the promoter resides within the proximal 4.5 kb of the MLC1v DNA fragment, whereas key elements regulating chamber‐specific expression are present in the proximal‐most 1.5 kb. Finally, we demonstrate how cardiac and craniofacial muscle expression of the MLC1v promoter can be used to diagnose mutant phenotypes in living embryos, using the injection of RNA encoding a Tbx1‐engrailed repressor–fusion protein as an example. Development Dynamics 232:1003–1012, 2005.

Hypoxic Regulation of Hand1 Controls the Fetal-Neonatal Switch in Cardiac Metabolism

This study reveals a novel pathway that responds to hypoxia and modulates energy metabolism by cardiomyocytes in the mouse heart, thereby determining oxygen consumption.

Targeted cell‐ablation in Xenopus embryos using the conditional, toxic viral protein M2(H37A)

Harnessing toxic proteins to destroy selective cells in an embryo is an attractive method for exploring details of cell fate and cell–cell interdependency. However, no existing “suicide gene” system has proved suitable for aquatic vertebrates. We use the M2(H37A) toxic ion channel of the influenza‐A virus to induce cell‐ablations in Xenopus laevis. M2(H37A) RNA injected into blastomeres of early stage embryos causes death of their progeny by late‐blastula stages. Moreover, M2(H37A) toxicity can be controlled using the M2 inhibitor rimantadine. We have tested the ablation system using transgenesis to target M2(H37A) expression to selected cells in the embryo. Using the myocardial MLC2 promoter, M2(H37A)‐mediated cell death causes dramatic loss of cardiac structure and function by stage 39. With the LURP1 promoter, we induce cell‐ablations of macrophages. These experiments demonstrate the effectiveness of M2(H37A)‐ablation in Xenopus and its utility in monitoring the progression of developmental abnormalities during targeted cell death experiments. Developmental Dynamics 236:2159–2171, 2007.

Xenopus Hand2 expression marks anterior vascular progenitors but not the developing heart.

We have isolated cDNAs encoding the bHLH protein Hand2 in the amphibian Xenopus laevis and analysed Hand2 expression in early development from the onset of gastrulation to feeding tadpole stages. XHand2 is expressed in the branchial arch mesenchyme and also in small bilateral populations of cells in the anterior, ventrolateral region of early tailbud embryos. At later stages, these punctate Hand2‐expressing cells are located at the sites of the forming common cardinal veins, suggesting that they may constitute progenitors of vascular smooth muscle cells. Other Hand2‐expressing cells are also associated with further components of the forming anterior vasculature but are not detected in mature blood vessels. Interestingly, no myocardial expression of XHand2 can be detected in the developing tadpole heart, in marked contrast to results obtained with chick and mouse embryos.

05-P008 Genome-wide Identification of Nkx2-5-binding sites in the embryonic heart

In this study we have performed genome-wide analysis to identify Nkx2-5 binding regions using mid-gestation mouse hearts to gain insight into the molecular mechanisms by which Nkx2-5 contributes to the correct development of the heart. Nkx2-5 and SRF invivo binding sites were mapped using ChIPchip assays and analysis of the ChIP-chip data has identified 600 and 858 SRF and Nkx2-5-binding sites, respectively. Independent ChIP validation of 20 randomly selected binding loci shows that 18 sites have greater than 2.5 and up to 30-fold enrichment in Nkx2-5 binding. In addition many known Nkx2-5 targets were identified for e.g. Nppa, Myocd, Actc1, Ankrd1, Calr, Smpx, Slc8a1, Mov10l1 and Cx40. Based on this evidence we predict that 85% of the binding sites identified in the Nkx2-5 ChIP are true positives. Furthermore, we found Nkx2-5 binding sites were significantly over-represented in the Nkx2-5-bound-regions. To identify direct gene targets of Nkx2-5 we have correlated the global binding of Nkx2-5 with global Nkx2-5 dependent expression analysis using a hypomorphic Nkx2-5IRES/CRE/+GFP mouse model that expresses reduced levels of Nkx2-5 and displays cardiac phenotypes observed in CHD (Prall, 2007). Using this approach we have identified 73 genes that are directly regulated by Nkx2-5 invivo and includes genes known to be important in cardiogenesis, such as Mov10l1 (Csm), Cited2, Csrp3 (MLP), Smpx (chisel), Smpd1 (Bob), Lrrc10 and many of unknown function.