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Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Matrigel/rat tail collagen I hydrogel was used as control. Resulting constructs were cultivated either in serum-free medium or in endothelial growth medium-2 serving as control. Endothelial cell networks were quantified, tested for lumen formation, and interaction of HUVECs and hASCs. Tube diameter was slightly larger in constructs containing human collagen I compared to Matrigel/rat tail collagen I constructs under serum-free conditions. All other network parameters were mostly similar. Thereby, the feasibility of generating 3D endothelial cell networks under serum-free culture conditions in human collagen I as hydrogel was demonstrated. In summary, the presented achievements pave the way for the generation of clinical applicable constructs.

The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present study finds prostaglandin E2 (PGE2) blood levels elevated in COVID-19 patients with positive correlation with disease severity. SARS-CoV-2 induces PGE2 generation and secretion in infected lung epithelial cells by upregulating cyclo-oxygenase (COX)-2 and reducing the PG-degrading enzyme 15-hydroxyprostaglandin-dehydrogenase. Also living human-lung-precision-slices infected with SARS-CoV-2 display upregulated COX-2. PGE2 in serum of COVID-19 patients lowers the expression of Paired-Box-Protein-Pax-5 (PAX5), a master regulator of B-cell survival, proliferation and differentiation, in both human and mouse pre-B-cells, while the PGE2 inhibitor taxifolin directly reduces SARS-CoV-2-induced PGE2 production and attenuates viral replication. Risk-factors for severe disease courses, i.e. older age, male sex and air pollution are associated with higher PGE2 production and lower PAX5 expression in pre-B-cells. Since PGE2 acts broadly immunosuppressive its elevation might reduce the early anti-viral defense and its inhibition may therefore reduce severe disease courses.

Tissue engineering utilizes an interdisciplinary approach to generate constructs for the treatment and repair of diseased organs. Generation of small vessels as vascular grafts or as envisioned central vessel for vascularized constructs is still a challenge. Here, the decellularization of porcine vessels by a non-detergent based protocol was developed and investigated. Perfusion-decellularization with sodium hydroxide solution resulted in removal of cellular material throughout the whole length of the vessel while preserving structural and mechanical integrity. A re-endothelialization of the retrieved matrix with human umbilical vein endothelial cells and cardiac endothelial cells was achieved through rotation-based seeding employing a custom-made bioreactor. A confluent monolayer was detected on the entire luminal surface. Thus, a non-detergent-based decellularization method allowing the re-endothelialization of the luminal surface was developed in this study, thereby paving the way for future implementation of the resulting construct as vascular graft or as central vessel for tissue engineered constructs in need of a perfusion system with readily available anastomosis sites.

In natural tissues, the nutrition of cells and removal of waste products is facilitated by a dense capillary network which is generated during development. This perfusion system is also indispensable for tissue formation in vitro. Nutrition depending solely on diffusion is not sufficient to generate tissues of clinically relevant dimensions, which is a core aim in tissue engineering research. In this study, the establishment of a vascular network was investigated in a self-assembling approach employing endothelial and mural cells. The process of vascularization was analyzed in constructs based on a carrier matrix of decellularized porcine small intestinal submucosa (SIS). A three-dimensional hydrogel containing Matrigel™, collagen, and respective cells was casted on top of the SIS. Various types of human endothelial cells (hECs), e.g. HUVECs, cardiac tissue ECs (hCECs), pulmonary artery ECs (hPAECs), and iPSC-derived ECs, were co-cultured with human adipose tissue-derived stromal cells (hASCs) within the hydrogel. Analyzed hECs were able to self-assemble and form three-dimensional networks harboring small caliber lumens within the hydrogel constructs in the presence of hASCs as supporting cells. Additionally, microvessel assembling required exogenous growth factor supplementation. This study demonstrates the development of stable vascularized hydrogels applying hASCs as mural cells in combination with various types of hECs, paving the way for the generation of clinically applicable tissue engineered constructs.

Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. Statement of Significance Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis.

Cardiovascular disorders and associated morbidities remain the leading cause of premature death worldwide. Since the regeneration of diseased hearts is very limited and the insufficient supply of donor organs persists, hopes rely on new therapies for heart repair. Reviving the proliferation of endogenous cardiomyocytes (CMs) or the administration of adult stem cells to the heart was of limited curative success to date. Thus, the administration of in vitro generated CMs is under investigation to replenish loss of functional heart muscle tissue. This requires a sustainable source of CMs. Induced pluripotent stem cells (iPSC) have raised hopes for developing autologous cell therapies. To serve for heart repair, efficient and safe iPSC differentiation protocols for CMs production are required. iPSC differentiation into CMs and even functional subtypes was indeed achieved in recent years, either by the ectopic expression of cardiac transcription factors or the supplementation of chemical pathway modulators. An alternative approach aims at the direct transdifferentiation of fibroblasts, which are present in the interstitial tissue of many organs, into functional lineage-specific cell types. As a result the formation of induced cardiomyocyte-like cells (iCMs) by the ectopic expression of specific transcription factors combinations has been demonstrated in vitro and in vivo. This is an important proof-of-concept that the intermediate state of iPSC induction is dispensable. However, most of the early experiments were conducted in mice and translation to more relevant large animal models and subsequently to the clinic are challenging. Progress, drawbacks, and perspectives in this field will be discussed.

The ultimate goal of tissue engineering is the generation of implants similar to native tissue. Thus, it is essential to utilize physiological stimuli to improve the quality of engineered constructs. Numerous publications reported that mechanical stimulation of small-sized, non-perfusable, tissue engineered cardiac constructs leads to a maturation of immature cardiomyocytes like neonatal rat cardiomyocytes or induced pluripotent stem cells/embryonic stem cells derived self-contracting cells. The aim of this study was to investigate the impact of mechanical stimulation and perfusion on the maturation process of large-scale (2.5 × 4.5 cm), implantable cardiac patches based on decellularized porcine small intestinal submucosa (SIS) or Biological Vascularized Matrix (BioVaM) and a 3-dimensional construct containing neonatal rat heart cells. Application of cyclic mechanical stretch improved contractile function, cardiomyocyte alignment along the stretch axis and gene expression of cardiomyocyte markers. The development of a complex network formed by endothelial cells within the cardiac construct was enhanced by cyclic stretch. Finally, the utilization of BioVaM enabled the perfusion of the matrix during stimulation, augmenting the beneficial influence of cyclic stretch. Thus, this study demonstrates the maturation of cardiac constructs with clinically relevant dimensions by the application of cyclic mechanical stretch and perfusion of the starter matrix. Statement of significance Considering the poor endogenous regeneration of the heart, engineering of bioartificial cardiac tissue for the replacement of infarcted myocardium is an exciting strategy. Most techniques for the generation of cardiac tissue result in relative small-sized constructs insufficient for clinical applications. Another issue is to achieve cardiomyocytes and tissue maturation in culture. Here we report, for the first time, the effect of mechanical stimulation and simultaneous perfusion on the maturation of cardiac constructs of clinical relevant dimensions, which are based on a perfusable starter matrix derived from porcine small intestine. In response to these stimuli superior organization of cardiomyocytes and vascular networks was observed in contrast to untreated controls. The study provides substantial progress towards the generation of implantable cardiac patches.

In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

Introduction A major obstacle in tissue engineering is to create a surgically implantable tissue with long-term viability. Several promising techniques have focused on biological vascularized matrices (BioVaM) with preserved vascular pedicles in the porcine model. However, the handling of this model is time-consuming and expensive. Therefore, our aim was to establish a BioVaM in the rat. Materials and Methods Small bowel segments of Sprague-Dawley rats were isolated and perfused via cannulation of the superior mesenteric artery and the portal vein. All cellular matrix components were removed by sequential treatment with sodium dodecyl sulfate, sodium deoxycholate, and DNase. Quality of decellularization was investigated by histology and potential residual DNA by spectrophotometry. Primary endothelial cells (ECs) isolated from the major vessels of Sprague-Dawley rats. Cells were labeled with fluorescent cell tracker and injected into the vascular pedicles of the matrix. Attachment of ECs was assessed using fluorescence microscopy of the whole mount. Results Decellularized matrix demonstrated the absence of cellular components but conserved matrix architecture as determined by immune fluorescent, pentachrome, and hematoxylin and eosin stains. DNA content was reduced by more than 99%. ECs were characterized by specific staining against endothelial nitric oxide synthase and von Willebrand factor; when injected, ECs attached along the vessel walls including the capillaries of the intestinal wall. Conclusions Rat small bowel segments harvested with intact vascular pedicles and associated vascular network can be successfully decellularized and re-endothelialized ex vivo. This model is an inexpensive and easy to handle alternative and appears to be a promising approach for establishing vascularized tissue constructs.

Nodal proteins are secreted signaling factors of the transforming growth factor β (TGFβ) family with essential roles in embryonic development in vertebrates. Mutations affecting the Nodal factors have severe consequences in mammals and fish. Furthermore, increased Nodal levels have been associated with melanoma tumor progression. Like other TGFβ-related proteins, Nodal factors consist of a pro-domain and a mature domain. The pro-domain of mouse Nodal protein stabilizes its precursor. However, the mechanisms by which the pro-domains exert their activities are unknown. Here, we characterize the zebrafish Nodal-related factor Cyclops (Cyc) and find unexpected functions for the pro-domain in regulating Cyc activity. We identified a lysosome-targeting region in the Cyc pro-domain that destabilizes the precursor and restricts Cyc activity, revealing the molecular basis for the short-range signaling activities of Cyc. We show that both the pro- and mature-domains of Cyc regulate its stability. We also characterize a mutation in the pro-domain of human NODAL (hNODAL) that underlies congenital heterotaxia. Heterologous expression of mutant hNODAL increases expression of Nodal-response genes. Our studies reveal unexpected roles for the pro-domain of the Nodal factors and provide a possible mechanism for familial heterotaxia.

Regenerative medicine, including preclinical studies in large animal models and tissue engineering approaches as well as innovative assays for drug discovery, will require the constant supply of hPSC-derived cardiomyocytes and other functional progenies. Respective cell production processes must be robust, economically viable and ultimately GMP-compliant. Recent research has enabled transition of lab scale protocols for hPSC expansion and cardiomyogenic differentiation towards more controlled processing in industry-compatible culture platforms. Here, advanced strategies for the cultivation and differentiation of hPSCs will be reviewed by focusing on stirred bioreactor-based techniques for process upscaling. We will discuss how cardiomyocyte mass production might benefit from recent findings such as cell expansion at the cardiovascular progenitor state. Finally, remaining challenges will be highlighted, specifically regarding three dimensional (3D) hPSC suspension culture and critical safety issues ahead of clinical translation.

Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20 % FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left–right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger–Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left–right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.

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.

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.

cNkx2-8 represents a novel member of the NK2-family transcription factors. The gene contains three highly conserved regions, the TN-, NK2-, and homeodomains which are diagnostic for this group of proteins. cNkx2-8 is expressed during chick embryogenesis in ventral foregut endoderm, myocardial mesoderm, epithelium of the branchial arches and the dorsal mesocardium. While cNkx2-8 expression partially overlaps with other NK genes, such as Nkx2-5 and Nkx2-3, its onset and aspects of its expression domains are specific. Thus, structural data and the expression profile suggest that cNkx2-8 constitutes a new homeobox protein which may cooperate with its known relatives in defining an antero-ventral field including the developing heart and pharyngeal endoderm.

Tissue engineering (TE) is an emerging interdisciplinary field aiming at the restoration or improvement of impaired tissue function. A combination of cells, scaffold materials, engineering methods, and biochemical and physiological factors is employed to generate the desired tissue substitute. Scaffolds often play a pivotal role in the engineering process supporting a three-dimensional tissue formation. The ideal scaffold should mimic the native extracellular environment providing mechanical and biological properties to allow cell attachment, migration, and differentiation, as well as remodeling by the host organism. The scaffold should be nonimmunogenic and should ideally be resorbed by the host over time, leaving behind only the regenerated tissue. More than 40 years ago, a preparation of the small intestine was introduced for the replacement of vascular structures. Since then the small intestinal submucosa (SIS) has gained a lot of interest in TE and subsequent clinical applications, as this material exhibits key features of a highly supportive scaffold. This review will focus on the general properties of the SIS and its applications in therapeutical approaches as well as in generating tissue substitutes in vitro. Furthermore, the main problem of TE, which is the insufficient nourishment of cells within three-dimensional, artificial tissues exceeding certain dimensions is addressed. To solve this issue the implementation of another small intestine-derived preparation, the biological vascularized matrix (BioVaM), could be a feasible option. The BioVaM comprises in addition to SIS the arterial and venous mesenteric pedicles and exhibits thereby a perfusable vessel bed that is preserved after decellularization.

The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases.

Taurine is the most abundant free amino acid in heart muscle and protects against heart failure. In the present study, the consequences of hereditary taurine deficiency on cardiac gene expression were examined in 2- and 15–16-month-old taurine transporter knockout (taut-/-) mice using a mouse-specific DNA microarray. This oligonucleotide-based microarray contains probes for 251 genes with relevance for heart function. Of these, 163 probes exhibited a reproducible hybridization signal and were analyzed. α-Actin type 1 mRNA levels were 70% lower in the heart of young and oldertaut-/-mice compared to wild-type controls. Interestingly, the hearts oftaut-/-mice showed a switch from α-actin 1 to α-actin 2 expression, as confirmed by real-time PCR and Western blot analysis. In addition, mRNA levels of biomarkers for pressure overload and hypertension were upregulated intaut-/-hearts, i.e., atrial natriuretic factor (+848%), brain natriuretic peptide (+90%), cardiac ankyrin repeat protein (+118%), and procollagen 1a1, 1a2 and 3a1 (+40% at least). These results point to a stress situation in the heart oftaut-/-mice under laboratory conditions, and it can be speculated thattaut-/-hearts may be even more susceptible to failure in the wild when under exogenous stress.

Popeye (Pop) genes are a novel gene family encoding putative transmembrane proteins predominantly present in striated and smooth muscle cells. In this study, a null mutation of Pop1 was generated by replacing the first coding exon of the Pop1 gene with the lacZ reporter gene. Homozygous mice lacking Pop1 were fertile and had a normal life span without any apparent phenotype. LacZ staining of tissues of heterozygous and homozygous Pop1-LacZ mice revealed strong expression in embryonic and fetal hearts. Pop1-LacZ was also expressed in the myotome and in myogenic progenitor cells within the limb and in smooth muscle cells of various organs. In the heart, Pop1-LacZ activity was downregulated postnatally in heterozygous mice but not in homozygous mice. Administration of the beta-adrenergic agonist isoproterenol led to a rapid increase in Pop1-LacZ activity in heterozygotes without induction at the transcriptional level, suggesting stabilization of the protein. No difference, however, was observed between homozygous and heterozygous mice in the ability to develop cardiac hypertrophy in response to isoproterenol. The capacity to regenerate skeletal muscle was tested after cardiotoxin injection into the hind limbs of hetero- and homozygous mice. In activated satellite cells of both genotypes, rapid activation of Pop1-LacZ expression was observed. In heterozygous animals, LacZ activity was only transiently elevated in muscle precursor cells undergoing fusion and in newly formed myotubes. In homozygotes, persistence of LacZ expression and a retarded ability to regenerate skeletal muscle were apparent, suggesting that Pop1 plays a role in muscle regeneration.

Heart disease is a leading cause of death and disability in the Western world and a major factor in rising health care costs. Despite great efforts and remarkable advances made over the past years, the molecular basis for heart development and the evolution of acquired heart disease are still poorly defined (Chien 2000; Srivastava and Olson 2000). Approximately 10% of spontaneous abortions and stillborns are caused by severe cardiac malformations (Hoffman 1995a, 1995b). In addition, congenital heart defects including aberrant cardiac outflow septation, valve formation, aortic stenosis or hypoplastic left ventricle are prevalent with a frequency of 8 cases in 1000 births. Understanding cardiac development in cellular and molecular terms, therefore, is of major clinical relevance. Key genes in cardiac development are also important components of regulatory circuits in the adult heart (Chien 2000). For example, cardiacrestricted transcription factors such as Nkx2.5 and GATA4 have been implicated, both in cardiac development and under pathophysiological conditions, in the adult heart. GATA4 and its interacting protein partner NFATc have been identified as important components of a signalling pathway involved in generating cardiac hypertrophy (Molkentin et al. 1998). Point mutations in human NKX2.5 (CSX) have been found to cause multiple congenital malformations including atrial and ventricular septal defects, Ebstein’s anomaly and other tricuspid valve abnormalities. NKX2.5 mutations are also responsible for familial ventricular arrhythmias in the adult (Schott et al. 1998; Benson et al. 1999). Thus, understanding cardiac development at the molecular level will result in the identification of genetic networks, which are active in various pathophysiological processes in the adult heart as well. For further reading on cardiac development the reader is referred to other recent reviews on these topics (Chen and Fishman 2000; Lough and Sugi 2000; Rosenthal and Xavier-Neto 2000; Srivastava and Olson 2000).

Members of the Cited family are nuclear transactivators which bind to the coactivators p300 and CBP. While Cited1 also binds to the TGFβ 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.

We identified a novel gene family in vertebrates which is preferentially expressed in developing and adult striated muscle. Three genes of the Popeye (POP) family were detected in human and mouse and two in chicken. Chromosomal mapping indicates that Pop1 and Pop3 genes are clustered on mouse chromosome 10, whereas Pop2 maps to mouse chromosome 16. We found evidence that POP1 and POP3 in chicken may also be linked and multiple transcript isoforms are generated from this locus. The POP genes encode proteins with three potential transmembrane domains that are conserved in all family members. Individual POP genes exhibit specific expression patterns during development and postnatally. Chicken POP3 and mouse Pop1 are first preferentially expressed in atrium and later also in the subepicardial compact layer of the ventricles. Chicken POP1 and mouse Pop2 are expressed in the entire heart except the outflow tract. All three Pop genes are expressed in heart and skeletal muscle of the adult mouse and lower in lung. Pop1 and Pop2 expression is upregulated in uterus of pregnant mice. Like the mouse genes, human POP genes are predominantly expressed in skeletal and cardiac muscle. The strong conservation of POP genes during evolution and their preferential expression in heart and skeletal muscle suggest that these novel proteins may have an important function in these tissues in vertebrates. (C) 2000 Academic Press.