Thomas Brand

Heart development: molecular insights into cardiac specification and early morphogenesis

The heart develops from two bilateral heart fields that are formed during early gastrulation. In recent years, signaling pathways that specify cardiac mesoderm have been extensively analyzed. In addition, a battery of transcription factors that regulate different aspects of cardiac morphogenesis and cytodifferentiation have been identified and characterized in model organisms. At the anterior pole, a secondary heart field is formed, which in its molecular make-up, appears to be similar to the primary heart field. The cardiac outflow tract and the right ventricle to a large extent are derivatives of this anterior heart field. Cardiac mesoderm receives positional information by which it is patterned along the three body axes. The molecular control of left-right axis development has received particular attention, and the underlying regulatory network begins to emerge. Cardiac chamber development involves the activation of a transcription program that is different from the one present in the primary heart field and regulates cardiac morphogenesis in a region-specific manner. This review also attempts to identify areas in which additional research is needed to fully understand early cardiac development.

BMP-2 induces ectopic expression of cardiac lineage markers and interferes with somite formation in chicken embryos

In Drosophila induction of the homeobox gene tinman and subsequent heart formation are dependent on dpp signaling from overlying ectoderm. In order to define vertebrate heart-inducing signals we screened for dpp-homologues expressed in HH stage 4 chicken embryos. The majority of transcripts were found to be BMP-2 among several other members of the BMP family. From embryonic HH stage 4 onwards cardiogenic mesoderm appeared to be in close contact to BMP-2 expressing cells which initially were present in lateral mesoderm and subsequently after headfold formation in the pharyngeal endoderm. In order to assess the role of BMP-2 for heart formation, gastrulating chick embryos in New culture were implanted with BMP-2 producing cells. BMP-2 implantation resulted in ectopic cardiac mesoderm specification. BMP-2 was able to induce Nkx2-5 expression ectopically within the anterior head domain, while GATA-4 was also induced more caudally. Cardiogenic induction by BMP-2, however remained incomplete, since neither Nkx2-8 nor the cardiac-restricted structural gene VMHC-1 became ectopically induced. BMP-2 expressing cells implanted adjacent to paraxial mesoderm resulted in impaired somite formation and blocked the expression of marker genes, such as paraxis, Pax-3, and the forkhead gene cFKH-1. These results suggest that BMP-2 is part of the complex of cardiogenic signals and is involved in the patterning of early mesoderm similar to the role of dpp in Drosophila.

BMP2 is required for early heart development during a distinct time period

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.

The homeobox gene it NKX3.2 is a target of left–right signalling and is expressed on opposite sides in chick and mouse embryos

Vertebrate internal organs display invariant left-right (L-R) asymmetry. A signalling cascade that sets up L-R asymmetry has recently been identified (reviewed in [1]). On the right side of Hensens node, activin represses Sonic hedgehog (Shh) expression and induces expression of the genes for the activin receptor (ActRIIa) and fibroblast growth factor-8 (FGF8) [2] [3]. On the left side, Shh induces nodal expression in lateral plate mesoderm (LPM); nodal in turn upregulates left-sided expression of the bicoid-like homeobox gene Pitx2 [4] [5] [6]. Here, we found that the homeobox gene NKX3.2 is asymmetrically expressed in the anterior left LPM and in head mesoderm in the chick embryo. Misexpression of the normally left-sided signals Nodal, Lefty2 and Shh on the right side, or ectopic application of retinoic acid (RA), resulted in upregulation of NKX3.2 contralateral to its normal expression in left LPM. Ectopic application of FGF8 on the left side blocked NKX3.2 expression, whereas the FGF receptor-1 (FGFR-1) antagonist SU5402, implanted on the right side, resulted in bilateral NKX3.2 expression in the LPM, suggesting that FGF8 is an important negative determinant of asymmetric NKX3.2 expression. NKX3.2 expression was also found to be asymmetric in the mouse LPM but, unlike in the chick, it was expressed in the right LPM. In the inversion of embryonic turning (inv) mouse mutant, which has aberrant L-R development, NKX3.2 was expressed predominantly on the left side. Thus, NKX3.2 transcripts accumulate on opposite sides of mouse and chick embryos although, in both the mouse and chick, NKX3.2 expression is controlled by the L-R signalling pathways.

Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos.

The proepicardium (PE) is a primarily extracardiac progenitor cell population that colonizes the embryonic heart and delivers the epicardium, the subepicardial and intramyocardial fibroblasts, and the coronary vessels. Recent data show that PE‐derived cells additionally play important regulatory roles in myocardial development and possibly in the normal morphogenesis of the heart. Developmental Dynamics 233, 2005. Research on the latter topics profits from the fact that loss‐of‐PE‐function can be experimentally induced in chick embryos. So far, two microsurgical techniques were used to produce such embryos: (1) blocking of PE cell transfer with pieces of the eggshell membrane, and (2) mechanical excision of PE. Both of these techniques, however, have their shortcomings. We have searched, therefore, for new techniques to eliminate the PE. Here, we show that loss‐of‐PE‐function can be induced by photoablation of the PE. Chick embryos were treated in ovo by means of a window in the eggshell at Hamburger and Hamilton (HH) stage 16 (iday 3). The pericardial coelom was opened, and the PE was externally stained with a 1% solution of Rose Bengal by means of a micropipette. Photoactivation of the dye was accomplished by illumination of the operation field with visible light. Examination on postoperative day 1 (iday 4, HH stages 19/20) disclosed complete removal of PE in every experimental embryo. On iday 9 (HH stages 33/34), the survival rate of experimental embryos was 35.7% (15 of 42). Development of the PE‐derivatives was compromised in the heart of every survivor. The abnormalities encompassed hydro‐ or hemopericardium, epicardium‐free areas with aneurysmatic outward bulging of the ventricular wall, thin myocardium, defects of the coronary vasculature, and abnormal tissue bridges between the ventricles and the pericardial wall. Our results show that photoablation of the PE is a powerful technique to induce long‐lasting loss‐of‐PE‐function in chick embryos. We have additionally obtained new data that suggest that the embryonic epicardium may make important contributions to the passive mechanics of the developing heart. Developmental Dynamics 233:1454–1463, 2005.

Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality‐associated cardiac anomalies. In animal models, a common read‐out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [ 2002 ] Dev. Biol. 246:407–417). To address this question further we used a reported loss‐of‐function approach to interfere with chick CFC expression (Schlange et al. [ 2001 ] Dev. Biol. 234:376–389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping. Developmental Dynamics 228:217–230, 2003.

Popeye domain containing gene 2 (Popdc2) is a myocyte-specific differentiation marker during chick heart development

The Popeye domain containing (popdc) genes constitute a novel gene family encoding proteins of the plasma membrane in muscle cells, with three N‐terminal transmembrane domains and a cytoplasmic carboxy terminus. In vertebrates, three members of the Popdc gene family have been described. However, in the chick system only two cDNAs, Popdc1 and Popdc3, have been cloned previously. By screening a chick expressed sequence tag database, we report here the identification of five alternatively spliced chick Popdc2 cDNAs with different carboxy termini. Northern blot analysis revealed expression of Popdc2 predominantly in the myocardium and weaker expression in skeletal muscle. By whole‐mount in situ hybridization, chick Popdc2 was first detected at Hamburger and Hamilton (HH) stage 7 within the anterior part of the heart fields. In the tubular heart, atrial and ventricular precursor cells stained positively for Popdc2. Weaker expression was observed in myocardium of the outflow tract and sinus venosus. By HH stage 18, the outer curvature myocardium was strongly stained, whereas expression in myocardium of the inner curvature was negligible. Popdc2 expression was absent from the endocardium and propepicardial organ. At HH stage 36, Popdc2 expression was confined to the compact layer myocardium. In addition to the heart, Popdc2 expression was also observed in the myotome and in the muscle‐forming fields of the limbs. Our results indicate that Popdc2 is highly expressed in the developing heart and may serve as a novel marker of myocardial differentiation in the chick embryo. Developmental Dynamics 229:695–702, 2004.

Expression analysis of the chicken homologue of CITED2 during early stages of embryonic development

Members of the Cited family are nuclear transactivators which bind to the coactivators p300 and CBP. While Cited1 also binds to the TGFbeta signal transducer Smad4, this has not been shown for Cited2. We isolated a chicken homologue of Cited2 from a HH stage 3-6 cDNA library and examined its expression pattern during early stages of embryonic development by whole-mount in situ hybridization. CITED2 expression is detectable in the epiblast as early as stage XI. From HH stage 2 onwards CITED2 is expressed in an anterior domain in the elongating primitive streak in cells which are fated to become heart. During gastrulation the expression pattern is highly dynamic and transiently displays left-right asymmetry with stronger expression on the right side. CITED2 expression appears at multiple sites of forming mesodermal structures. Most prominently, CITED2 is expressed in presomitic and lateral plate mesoderm, in the headfold (future forebrain), the head mesoderm, the pharyngeal floor, the ventral blood islands, somitomeres and the intermediate mesoderm which gives rise to the kidney anlagen.

Cardiac specific expression of Xenopus Popeye-1

Popeye genes code for putative transmembrane proteins that are predominantly expressed in heart and skeletal muscle. Here we report on the isolation and expression of a previously unknown Xenopus member of this family, Xenopus Popeye-1 (Xpop-1). Xpop-1 is 60-65% identical to other vertebrate Pop-1 genes at the protein level. Whole-mount in situ hybridization studies revealed a highly specific expression of Xpop-1 whose transcripts are restricted to the embryonic heart and become enriched in the forming ventricle. Interestingly, unlike other known vertebrate Popeye genes, Xpop-1 is exclusively expressed in cardiac tissue and absent from skeletal muscle.

Molecular genetics of muscle development andneuromuscular diseases: Kloster Irsee, Germany, September 26–October 1, 1999

The meeting held at the Irsee Convent in Southern Germany was organized by Hans‐Henning Arnold (Braunschweig), Renate Renkawitz‐Pohl (Marburg), Anna Starzinski‐Powitz (Frankfurt) and Bodo Christ (Freiburg). A total of 150 participants, 47 of whom were invited speakers, created an atmosphere of intense progress in a multifaceted field of research. This comprised somite patterning and determination of cell lineages as a paradigm of muscle precursor cell specification, cardiac myogenesis, signals involved in myogenesis, differentiation and the control of muscle‐specific gene expression, as well as the molecular genetics of myopathies and repair processes. Eric Olson (Dallas, TX) gave the keynote address on the transcriptional control of myogenesis.nn### More signals and complexity: the somitesnnThe paraxial mesoderm is the source of cells that can differentiate into skeletal muscle. It is the primary structure in the vertebrate body showing segmentation when the somites form in a periodic sequence of cranio‐caudal subdivisions from the presomitic mesoderm in mice and the segmental plate in birds. Three years ago, Pourquies group (Palmeirim et al ., 1997) presented the first experimental evidence for a periodic molecular event prefiguring the synchronous formation of subsequent somite pairs. They described the oscillating expression of c‐hairy1 in the segmental plate prior to somite formation. Such an oscillation had been postulated in theoretical models of somitogenesis. While an ortholog of c‐hairy1 in mouse has not been found yet, new oscillating genes have been described and attempts are being made to arrange them in a sequence of events. The most prominent gene with oscillating expression is lunatic fringe , an ortholog of Drosophila fringe , which modulates the Delta‐Notch pathway (O.Pourquie, Marseille, France; R.Johnson, Houston, TX). Mice homozygous for a targeted deletion in lunatic fringe lack caudal somites, and markers of a cranio‐caudal polarization such as uncx 4.1 are expressed irregularly (Evrard et al ., 1998; Pourquie, 1999). It appears that …