Defective heart chamber growth and myofibrillogenesis after knockout of adprhl1 gene function by targeted disruption of the ancestral catalytic active site.

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

Furin, a transcriptional target of NKX2-5, has an essential role in heart development and function.

The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart.

The cardiac-restricted protein ADP-ribosylhydrolase-like 1 is essential for heart chamber outgrowth and acts on muscle actin filament assembly.

Adprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5'-coding sequence of Xenopus adprhl1. Over-expression of full length (40kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity.

Sequential Binding of MEIS1 and NKX2-5 on the Popdc2 Gene: A Mechanism for Spatiotemporal Regulation of Enhancers during Cardiogenesis.

The homeobox transcription factors NKX2-5 and MEIS1 are essential for vertebrate heart development and normal physiology of the adult heart. We show that, during cardiac differentiation, the two transcription factors have partially overlapping expression patterns, with the result that as cardiac progenitors from the anterior heart field differentiate and migrate into the cardiac outflow tract, they sequentially experience high levels of MEIS1 and then increasing levels of NKX2-5. Using the Popdc2 gene as an example, we also show that a significant proportion of target genes for NKX2-5 contain a binding motif recognized by NKX2-5, which overlaps with a binding site for MEIS1. Binding of the two factors to such overlapping sites is mutually exclusive, and this provides a simple regulatory mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.

Hypoxic regulation of hand1 controls the fetal-neonatal switch in cardiac metabolism.

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.

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.

Pan-myocardial expression of Cre recombinase throughout mouse development.

Mouse-lines expressing Cre recombinase in a tissue-specific manner are a powerful tool in developmental biology. Here, we report that a 3 kb fragment of the Xenopus laevis myosin light-chain 2 (XMLC2) promoter drives Cre recombinase expression in a cardiac-restricted fashion in the mouse embryo. We have isolated two XMLC2-Cre lines that express recombinase exclusively within cardiomyocytes, from the onset of their differentiation in the cardiac crescent of the early embryo. Expression is maintained throughout the myocardium of the embryonic heart tube and subsequently the mature myocardium of the chambered heart. Recombinase activity is detected in all myocardial tissue, including the pulmonary veins. One XMLC2-Cre line shows uniform expression while the other only expresses recombinase in a mosaic fashion encompassing less than 50% of the myocardial cells. Both lines cause severe cardiac malformations when crossed to a conditional Tbx5 line, resulting in embryonic death at midgestation. Optical projection tomography reveals that the spectrum of developmental abnormalities includes a shortening of the outflow tract and its abnormal alignment, along with a dramatic reduction in trabeculation of the ventricular segment of the looping heart tube.

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.

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.

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.

Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos.

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.