Gary E. Lyons

Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem Cells: The Matrix Sandwich Method

Rationale: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. Objective: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. Methods and Results: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions: Dynamic extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Expression Patterns of the Four Nuclear Factor I Genes During Mouse Embryogenesis Indicate a Potential Role in Development

The nuclear factor I (NFI) family of site‐specific DNA‐binding proteins is required for both the cell‐type specific transcription of many viral and cellular genes and for the replication of adenovirus DNA. Although binding sites for NFI proteins within the promoters of several tissue‐specific genes have been shown to be essential for their expression, it is unclear which NFI gene products function in specific tissues during development. We have isolated cDNAs from all four murine NFI genes (gene designations Nfia, Nfib, Nfic, and Nfix), assessed the embryonic and postnatal expression patterns of the NFI genes, and determined the ability of specific NFI proteins to activate transcription from the NFI‐dependent mouse mammary tumor virus (MMTV) promoter. In adult mice, all four NFI genes are most highly expressed in lung, liver, heart, and other tissues but only weakly expressed in spleen and testis. The embryonic expression patterns of the NFI genes is complex, with NFI‐A transcripts appearing earliest—within 9 days postcoitum in the heart and developing brain. The four genes exhibit unique but overlapping patterns of expression during embryonic development, with high level expression of NFI‐A, NFI‐B, and NFI‐X transcripts in neocortex and extensive expression of the four genes in muscle, connective tissue, liver, and other organ systems. The four NFI gene products studied differ in their ability to activate expression of the NFI‐dependent MMTV promoter, with the NFI‐B protein being most active and the NFI‐A protein being least active. These data are discussed in the context of the developmental expression patterns of known NFI‐responsive genes. The differential activation of an NFI‐dependent promoter, together with the expression patterns observed for the four genes, indicate that the NFI proteins may play an important role in regulating tissue‐specific gene expression during mammalian embryogenesis. Dev. Dyn. 208:313–325, 1997.

Essential Role for NFI-C/CTF Transcription-Replication Factor in Tooth Root Development

ABSTRACT The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and Nfix. In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic−/− mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic−/− mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic−/− animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.

The Microwell Control of Embryoid Body Size in order to Regulate Cardiac Differentiation of Human Embryonic Stem Cells

The differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs) using embryoid bodies (EBs) is relatively inefficient and highly variable. Formation of EBs using standard enzymatic disaggregation techniques results in a wide range of sizes and geometries of EBs. Use of a 3-D cuboidal microwell system to culture hESCs in colonies of defined dimensions, 100-500 microm in lateral dimensions and 120 microm in depth, enabled formation of more uniform-sized EBs. The 300 microm microwells produced highest percentage of contracting EBs, but flow cytometry for myosin light chain 2A (MLC2a) expressing cells revealed a similar percentage (approximately 3%) of cardiomyocytes formed in EBs from 100 microm to 300 microm microwells. These data, and immunolabeling with anti-MF20 and MLC2a, suggest that the smaller EBs are less likely to form contracting EBs, but those contracting EBs are relatively enriched in cardiomyocytes compared to larger EB sizes where CMs make up a proportionately smaller fraction of the total cells. We conclude that microwell-engineered EB size regulates cardiogenesis and can be used for more efficient and reproducible formation of hESC-CMs needed for research and therapeutic applications.

Jumonji, a Nuclear Protein That Is Necessary for Normal Heart Development

Jumonji (jmj) was cloned in a gene trap screen to identify and mutagenize genes important for heart development. To investigate the role of jmj in heart development, we generated mice homozygous for the jmj mutation. The jmj homozygous mouse embryos showed heart malformations, including ventricular septal defect, noncompaction of the ventricular wall, double-outlet right ventricle, and dilated atria. The jmj mutants died soon after birth, apparently as a result of respiratory insufficiency caused by rib and sternum defects in addition to the heart defects. In situ hybridization analyses suggested that cardiomyocytes were differentiated but developmental regulation of chamber-specific genes was defective in fetal hearts. Expression of jmj was detected in the myocardium, especially in the interventricular septum, ventricular wall, and outflow tract, which correlated well with the locations of defects observed in the hearts of mutant mice. Homozygous embryos failed to express the jmj transcript in all tissues except in the nervous system. Confocal microscopic examination using anti-JMJ antibodies indicated that the JMJ protein was localized in the nuclei of cells transfected with jmj. These data demonstrate that JMJ is a nuclear protein, which is essential for normal heart development and function.

MIZ1, A NOVEL ZINC FINGER TRANSCRIPTION FACTOR THAT INTERACTS WITH MSX2 AND ENHANCES ITS AFFINITY FOR DNA

Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull. We demonstrated previously that individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h). The mutation behaves as a dominant positive in transgenic mice. The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted us to undertake a yeast two hybrid screen for Msx2-interacting proteins. Here we present a functional analysis of one such protein, designated Miz1 (Msx-interacting-zinc finger). Miz1 is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein, Nfi-1. Together these proteins define a new, highly conserved protein family. Analysis of Miz1 expression by Northern blot and in situ hybridization revealed a spatiotemporal pattern that overlaps that of Msx2. Further, Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor. Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter. The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype.

Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin.

Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling molecules. Although there has been extensive interest in the mechanism by which the closely-related Akt kinase participates in phosphatidylinositol 3-kinase-mediated signaling, comparatively little is known regarding the expression and function of Akt2. This manuscript is the first to describe Akt2 mRNA expression in the developing mouse and the activation of AKT2 by insulin. These studies demonstrate that Akt2 is especially abundant in brown fat and, to a lesser extent, skeletal muscle and liver, tissues which are highly insulin-responsive and play a role in glucose metabolism. Endogenous Akt2 expression also is upregulated in fully-differentiated C2C12 myotubes and 3T3-L1 adipocytes, suggesting that these murine cell lines represent useful in vitro models for studies of Akt2 function. We show that HA-tagged AKT2 is activated in response to insulin stimulation in vitro and that activation of AKT2 is not induced in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These data suggest that Akt2 expression is fundamental to the differentiated state of fat and muscle cells and that activation of AKT2 kinase by insulin is mediated through the phosphatidylinositol 3-kinase signaling pathway.

In situ analysis of the cardiac muscle gene program during embryogenesis.

During the last decade, gene expression in cardiac muscle of the developing embryo has been examined in situ with the use of nucleic acid probes and antibodies. With growth and maturation from a simple tube to a four-chambered organ, the myocardium undergoes a complex series of developmental changes in the temporal and spatial expression patterns of a number of structural and transcription factor genes. Studies of embryonic hearts suggest that different populations of cardiac myocytes might appear during development. These in vivo results are summarized and discussed in the context of the morphogenetic events that shape the myocardium.

Vertebrate heart development

This review summarizes recent studies of the cellular and molecular events involved in the determination and differentiation of cardiac myocytes in vertebrate embryos. Fate-mapping studies in mouse, chick, amphibian and zebrafish embryos suggest that cardiac muscle precursors are specified shortly before or at the time of gastrulation. Nuclear factors, such as dHAND, aryl hydrocarbon receptor, GATA-6, Nkx-2.3, growth arrest homeobox (Gax) and cardiac adriamycin responsive protein (CARP), which have recently been described as playing a role in the commitment and/or differentiation of cardiac myocytes are discussed.

Vertebrate homologs of tinman and bagpipe: Roles of the homeobox genes in cardiovascular development

In Drosophila, dorsal mesodermal specification is regulated by the homeobox genes tinman and bagpipe. Vertebrate homologs of tinman and bagpipe have been isolated in various species. Moreover, there are at least four different genes related to tinman in the vertebrate, which indicates that this gene has been duplicated during evolution. One of the murine homologs of tinman is the cardiac homeobox gene Csx or Nkx2.5. Gene targeting of Csx/Nkx2.5 showed that this gene is required for completion of the looping morphogenesis of the heart. However, it is not essential for the specification of the heart cell lineage. Early cardiac development might therefore be regulated by other genes, which may act either independently or in concert with Csx/Nkx2.5. Possible candidates might be other members of the NK2 class of homeobox proteins like Tix/Nkx2.6, Nkx2.3, nkx2.7, or cNkx2.8. Murine Tix/Nkx2.6 mRNA has been detected in the heart and pharyngeal endoderm (this study). Xenopus XNkx2.3 and chicken cNkx2.3 are expressed in the heart as well as in pharyngeal and gut endoderm. In contrast, murine Nkx2.3 is expressed in the gut and pharyngeal arches but not the heart. In zebrafish and chicken, two new NK-2 class homeoproteins, nkx2.7 and cNkx2.8, have been identified. Zebrafish nkx2.7 is expressed in both, the heart and pharyngeal endoderm. In the chicken, cNkx2.8 is expressed in the heart primordia and the primitive heart tube and becomes undetectable after looping. No murine homologs of nkx2.7 or cNkx2.8 have been found so far. The overlapping expression pattern of NK2 class homeobox genes in the heart and the pharynx may suggest a common origin of these two organs. In the Drosophila genome, the tinman gene is linked to another NK family gene named bagpipe. A murine homolog of bagpipe, Bax/Nkx3.1, is expressed in somites, blood vessels, and the male reproductive system during embryogenesis (this study), suggesting that this genes function may be relevant for the development of these organs. A bagpipe homolog in Xenopus, Xbap, is expressed in the gut masculature and a region of the facial cartilage during development. In this paper, we discuss molecular mechanisms of cardiovascular development with particular emphasis on roles of transcription factors.