Manabu Shirai

T-box 2, a mediator of Bmp-Smad signaling, induced hyaluronan synthase 2 and Tgfβ2 expression and endocardial cushion formation

During early heart development, Tbx2 gene expression is initiated in the cardiac crescent and then becomes restricted to the outflow tract and the atrioventricular region. We identified a Tbx2 regulatory region, enriched in multiple Smad sites, sufficient to reproduce Tbx2 expression patterns overlapping Bmp2 and Bmp4 gene activity in the heart. The role of Tbx2 in cardiogenesis was analyzed by using Cre-LoxP activated Tbx2 transgenic misexpression in chamber myocardium. Ventricular Tbx2 misexpression exhibited an abnormally narrow chamber lumen owing to the expansion of Hyaluronan synthase 2 expression in the ECM or cardiac jelly and the appearance of the endocardial cushions (ECs). Excessive Tbx2 also induced Tgfβ2, which coincided with the outgrowth epithelial-mesenchymal transformed cells in ventricular and atrial tissues modifying cardiomyocyte identity from chamber type to non-chamber type. Tbx2, a central intermediary of Bmp-Smad signaling, has a central part in directing Has2 and Tgfβ2 expression, facilitating EC formation.

The Polycomb-group gene Rae28 sustains Nkx2.5/Csx expression and is essential for cardiac morphogenesis

The Polycomb-group (PcG) gene Rae28 is a mammalian homologue of the Drosophila gene polyhomeotic. PcG genes are known to maintain transcription states, once initiated, probably by regulating chromatin structure. Since homozygous Rae28-deficient (Rae28(-/-)) mice displayed cardiac anomalies similar to congenital heart diseases in humans, we examined the role of Rae28 in cardiac morphogenesis at the molecular level. In Rae28(-/-) embryos, expression of the cardiac selector gene Nkx2.5/Csx (Nkx2.5) was initiated properly but was not sufficiently sustained later in development. This impaired expression of Nkx2.5 in the maintenance phase proved to have a crucial effect on cardiac morphogenesis, as demonstrated by the results of a genetic complementation experiment in which the cardiac anomalies were suppressed by overexpression of human NKX2.5/CSX1 in Rae28(-/-) embryos. Ubiquitous expression of exogenous Rae28 likewise restored the impaired Nkx2.5 expression in Rae28(-/-) embryos, further supporting the notion that Rae28 sustains Nkx2.5 expression in cardiomyocytes. Thus, our data show that a mammalian PcG gene can play a key role in organogenesis by helping to maintain the expression of a selector gene.

The Cellular Prion Protein Identifies Bipotential Cardiomyogenic Progenitors

Rationale: The paucity of specific surface markers for cardiomyocytes and their progenitors has impeded the development of embryonic or pluripotent stem cell–based transplantation therapy. Identification of relevant surface markers may also enhance our understanding of the mechanisms underlying differentiation. Objective: Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors. Methods and Results: Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential. Conclusions: PrP+ cells from beating embryoid bodies (EBs) frequently included nascent Myhc+ cardiomyocytes. Cultured PrP+ cells further differentiated, giving rise to cardiac troponin I+ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR)&agr;, enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP+PDGFR&agr;+ (PRa) cells. The Myhc− PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc+, whereas they were Myhc− at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc− PRa cell–derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.

Myocardium-derived angiopoietin-1 is essential for coronary vein formation in the developing heart

The origin and developmental mechanisms underlying coronary vessels are not fully elucidated. Here we show that myocardium-derived angiopoietin-1 (Ang1) is essential for coronary vein formation in the developing heart. Cardiomyocyte-specific Ang1 deletion results in defective formation of the subepicardial coronary veins, but had no significant effect on the formation of intramyocardial coronary arteries. The endothelial cells (ECs) of the sinus venosus (SV) are heterogeneous population, composed of APJ-positive and APJ-negative ECs. Among these, the APJ-negative ECs migrate from the SV into the atrial and ventricular myocardium in Ang1-dependent manner. In addition, Ang1 may positively regulate venous differentiation of the subepicardial APJ-negative ECs in the heart. Consistently, in vitro experiments show that Ang1 indeed promotes venous differentiation of the immature ECs. Collectively, our results indicate that myocardial Ang1 positively regulates coronary vein formation presumably by promoting the proliferation, migration and differentiation of immature ECs derived from the SV.

RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation.

The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast‐to‐myotube transition. Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12. RNA‐binding proteins bind to specific target RNA sequences and regulate gene expression in a post‐transcriptional manner. This study demonstrates that RNA‐binding motif protein 24 (Rbm24) interacts with the 3′‐untranslated region of myogenin mRNA and affects its half‐life in C2C12 myogenesis. Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis. In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation. Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24‐knockdown cells. Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells. Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin‐dependent post‐transcriptional regulatory pathway.

A Gene Trap Strategy for Identifying the Gene Expressed in the Embryonic Nervous System

Abstract An efficient gene trap strategy was devised for identifying the genes that are expressed in the mouse developing nervous system. Mouse embryonic stem (ES) cell lines that carried independent integrations of a gene trap vector, pSneolNlacZA, were allowed to differentiate in a suspension culture system. To select cells containing neurons, astrocytes or neuronglia precursors, cell lines were immunohistochemically examined with antibodies against neuron-specific proteins (neurofilament protein 150 kD and microtubule associated protein 2), glial fibrillary acidic protein or nestin. Three cell clones (GT3-8, 11 and 12) were immunoreactive to either of the antibodies employed and at the same time positive for &bgr;-galactosidase activity. When chimeric embryos were generated by the use of the above 3 cell lines, some cells in their nervous system showed X-gal staining. Thus the major advantage of the present gene trap method lies in its prescreening step of manipulated ES cells prior to generation of chimeric animals. This method holds promise as a useful tool for investigating the genes involved in the development of the nervous system.

Expression of vinexin α in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal

Vinexin, a recently identified cytoskeletal protein, contains three SH3 domains and plays important roles in regulation of cytoskeletal organization and signal transduction. Using whole-mount in situ hybridization, we showed here that expression of vinexin alpha, the longer vinexin transcript, is strictly regulated, although the shorter transcript, vinexin beta, is expressed almost ubiquitously during embryonic development in mice. Expression of vinexin alpha was limited to within part of the eye and heart in 10.5 dpc embryos. Analysis of cryosections of 10.5 dpc embryos showed that vinexin alpha was expressed in a dorsal half of the retinal pigment epithelium and in the outflow tract and atrioventricular canal of the heart. Furthermore, we also found that vinexin alpha was expressed in the gonad and in a ventral part of the pons of 12.5 dpc embryos. These results indicated that the expression of vinexin alpha is strictly regulated in a temporally and spatially restricted manner.

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.

Differentiation of Pharyngeal Endoderm from Mouse Embryonic Stem Cell

Embryonic stem cells are considered to be a good in vitro tool to study the induction of various cell types including cardiomyocytes; however, induction of the pharyngeal endoderm (PE), the underlying heart-forming region, in vivo has been scarcely reported. In the present study, we found that many PE-related genes, such as Paxl, Pax9, Sixl, and Tbxl, were up-regulated in cardiomyocyte-rich embryoid bodies (EBs). The third pouch-related genes including Hoxa3, Foxn1, and Aire, which are crucial for thymus development and function, were also detected in later stages. Nkx2.5, a cardiac transcription factor gene, is known to be transiently expressed in the PE. By crossing Nkx2.5-Cre mice with Cre-dependent EGFP reporter mice, we found that Nkx2.5(+) lineage exclusively contributed to thymic epithelial cell development, followed by thymus development. Gene expression analysis using Nkx2.5-EGFP ES cells also revealed that PE-related mRNAs were specifically enriched in the transiently appearing E-cadherin(+)Nkx2.5(+) cell fraction. Interestingly, the EB-derived cells were found capable of supporting T-cell differentiation to CD4 or CD8 double-positive cells in a reaggregation organ culture in vitro. Our results suggest that EBs contain cells that resemble third pharyngeal pouch endoderm and confer a thymus-like microenvironment.

Overexpression of Polycomb-Group Gene rae28 in Cardiomyocytes Does Not Complement Abnormal Cardiac Morphogenesis in Mice Lacking rae28 But Causes Dilated Cardiomyopathy

The Polycomb-group genes (PcG) are widely conserved from Drosophila to mammals and are required for maintaining positional information during development. The rae28 gene (rae28) is a member of the mouse PcG. Mice deficient in rae28 (rae28−/−) demonstrated that rae28 has a role not only in anteroposterior patterning but also in cardiac morphogenesis. In this study we generated transgenic mice with ubiquitous or cardiomyocyte-specific exogenous rae28 expression. Genetic complementation experiments with these transgenic mice showed that ubiquitous expression of rae28 could reverse the cardiac anomalies in rae28−/−, whereas cardiomyocyte-specific expression of rae28 could not, suggesting that rae28 is involved in cardiac morphogenesis through a noncardiomyocyte pathway. Interestingly, however, cardiomyocyte-specific overexpression of rae28 caused dilated cardiomyopathy, which was associated with cardiomyocyte apoptosis, abnormal myofibrils, and severe heart failure. Cardiac expression of rae28 was predominant in the early embryonic stage, whereas that of the other PcG members was relatively constitutive. Because rae28 forms multimeric complexes with other PcG proteins in the nucleus, it is presumed that constitutive cardiomyocyte-specific rae28 overexpression impaired authentic PcG functions in the heart. rae28-induced dilated cardiomyopathy may thus provide a clue for clarifying the direct role of PcG in the maintenance of cardiomyocytes.