Chip/Ldb1 interacts with Tailup/islet1 to regulate cardiac gene expression in Drosophila.

The LIM-homeodomain transcription factor Tailup (Tup) is a component of the complex cardiac transcriptional network governing specification and differentiation of cardiac cells in Drosophila. LIM-domain containing factors are known to interact with the adaptor molecule Chip/Ldb1 to form higher order protein complexes to regulate gene expression thereby determining a cell's developmental fate. However, with respect to Drosophila heart development, it has not been investigated yet, whether Chip and tup interact to regulate the generation of different cardiac cell types. Here we show that Chip is required for normal heart development and that it interacts with tup in this context. Particularly the number of Odd skipped-expressing pericardial cells depends on balanced amounts of Chip and Tup. Data from luciferase assays using Hand- and even-skipped reporter constructs in Drosophila S2 cells indicate that Chip and Tup act as a tetrameric complex on the regulatory regions of Hand and even-skipped (eve). Finally we have identified and verified five Tup binding sites in the eve mesodermal enhancer, which adds Tup as novel factor to directly regulate eve expression. Taken together this study provides novel findings regarding cardiac gene expression regulation in Drosophila.

Nipped-A regulates intestinal stem cell proliferation in Drosophila.

Adult stem cells uphold a delicate balance between quiescent and active states, a deregulation of which can lead to age-associated diseases such as cancer. In Drosophila, intestinal stem cell (ISC) proliferation is tightly regulated and mis-regulation is detrimental to intestinal homeostasis. Various factors are known to govern ISC behavior; however, transcriptional changes in ISCs during aging are still unclear. RNA sequencing of young and old ISCs newly identified Nipped-A, a subunit of histone acetyltransferase complexes, as a regulator of ISC proliferation that is upregulated in old ISCs. We show that Nipped-A is required for maintaining the proliferative capacity of ISCs during aging and in response to tissue-damaging or tumorigenic stimuli. Interestingly, Drosophila Myc cannot compensate for the effect of the loss of Nipped-A on ISC proliferation. Nipped-A seems to be a superordinate regulator of ISC proliferation, possibly by coordinating different processes including modifying the chromatin landscape of ISCs and progenitors.

Isolating intestinal stem cells from adult Drosophila midguts by FACS to study stem cell behavior during aging.

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells' (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

The Iroquois complex is required in the dorsal mesoderm to ensure normal heart development in Drosophila.

Drosophila heart development is an invaluable system to study the orchestrated action of numerous factors that govern cardiogenesis. Cardiac progenitors arise within specific dorsal mesodermal regions that are under the influence of temporally coordinated actions of multiple signaling pathways. The Drosophila Iroquois complex (Iro-C) consists of the three homeobox transcription factors araucan (ara), caupolican (caup) and mirror (mirr). The Iro-C has been shown to be involved in tissue patterning leading to the differentiation of specific structures, such as the lateral notum and dorsal head structures and in establishing the dorsal-ventral border of the eye. A function for Iro-C in cardiogenesis has not been investigated yet. Our data demonstrate that loss of the whole Iro complex, as well as loss of either ara/caup or mirr only, affect heart development in Drosophila. Furthermore, the data indicate that the GATA factor Pannier requires the presence of Iro-C to function in cardiogenesis. Furthermore, a detailed expression pattern analysis of the members of the Iro-C revealed the presence of a possibly novel subpopulation of Even-skipped expressing pericardial cells and seven pairs of heart-associated cells that have not been described before. Taken together, this work introduces Iro-C as a new set of transcription factors that are required for normal development of the heart. As the members of the Iro-C may function, at least partly, as competence factors in the dorsal mesoderm, our results are fundamental for future studies aiming to decipher the regulatory interactions between factors that determine different cell fates in the dorsal mesoderm.

Islet1-expressing cardiac progenitor cells: a comparison across species.

Adult mammalian cardiac stem cells express the LIM-homeodomain transcription factor Islet1 (Isl1). They are considered remnants of Isl1-positive embryonic cardiac progenitor cells. During amniote heart development, Isl1-positive progenitor cells give rise mainly to the outflow tract, the right ventricle, and parts of the atria. This led to the hypothesis that the development of the right ventricle of the amniote heart depends on the recruitment of additional cells to the primary heart tube. The region from which these additional, Isl1-positive cells originate is called second heart field, as opposed to the first heart field whose cells form the primary heart tube. Here, we review the available data about Isl1 in different species, demonstrating that Isl1 is an important component of the core transcription factor network driving early cardiogenesis in animals of the two clades, deuterostomes, and protostomes. The data support the view of a single cardiac progenitor cell population that includes Isl1-expressing cells and which differentiates into the various cardiac lineages during embryonic development in vertebrates but not in other phyla of the animal kingdom.

A role for Drosophila Wnt-4 in heart development.

In vertebrates, different Wnt-signaling pathways are required in a temporally coordinated manner to promote cardiogenesis. In Drosophila, wingless holds an essential role in heart development. Among the known Drosophila Wnts is DWnt4, the function of which has been studied in various developmental processes except for heart development. We re-evaluated the expression pattern of DWnt4 during embryogenesis and show that transcripts are not restricted to the dorsal ectoderm but are also present in the cardiogenic mesoderm. Moreover, we detect DWnt4 mRNA transcripts in myocardial cells by stage 16. The heart phenotype in DWnt4 mutant embryos is characterized by various degrees of disrupted expression of different cardiac markers. Overexpression of Dwnt4 also affects heart marker expression, which can be partially rescued by simultaneous inhibition of PKC. Our data reveal a role for DWnt4 in cardiogenesis; however, integration of DWnt4 with other known signaling pathways that function in heart development still awaits further investigation.

Saving hearts through basic research.

Despite the recent advances in molecular medicine and health care, cardiovascular diseases are still the leading cause of morbidity and mortality throughout the world. In 2006, nearly every other death in Germany resulted from disease of the circulatory system, and congenital heart diseases are thought to account for a high number of stillbirths and spontaneous abortions. Remarkable progress in basic research over the past decades has improved our understanding of the molecular mechanisms that govern a cardiac fate and has helped to establish cell-based therapeutic approaches to improve the course of cardiovascular diseases.

The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis.

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. We have analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, we believe it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate.

Measuring CamKII activity in Xenopus embryos as a read-out for non-canonical Wnt signaling.

It has been known for quite some time that not all members of the Wnt family induce the formation of a secondary body axis when ectopically expressed in Xenopus embryos. An ingenious hypothesis led to the discovery that some Wnt ligands have the capacity to elicit intracellular Ca2+ signaling. This finding has been studied in more detail in the past years, which has revealed an intriguing complexity of Wnt signaling. The significance of a Wnt-induced Ca(2+)-mediated pathway during development has been demonstrated in various model systems so far and includes processes such as dorsal-ventral patterning, regulation of the canonical Wnt/beta-catenin signaling pathway, tumor formation, bone formation, and regulation of epithelial-mesenchymal transitions. Here we describe two assays to measure the activation of the Ca2+/calmodulin-dependent kinase (CamK)-II, a Ca(2+)-sensitive molecule described as a mediator of a non-canonical Wnt signaling pathway.

Dorsal axis duplication as a functional readout for Wnt activity.

The easy accessibility, distinctive features of early cleavage stage embryos and simple manipulation methods make Xenopus embryos an ideal model organism to study gene function and deciphering signaling pathways. For many years, investigators have analyzed putative dorsalizing factors by their ability to induce secondary dorsal structures when misexpressed in early Xenopus embryos. This assay, among others, has contributed substantially to our knowledge about Wnt signaling pathways and is still the assay of choice to quickly determine whether a factor acts positively or negatively in the Wnt signaling pathway. This chapter describes two experimental approaches to determine canonical Wnt signaling: induction of a secondary axis and analyses of target gene expression.

DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of cardiac identity in first heart field derived cells.

Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.

The amphibian second heart field: Xenopus islet-1 is required for cardiovascular development.

Islet-1 is a LIM-homeodomain transcription factor that has been defined to label cardiac progenitor cells of the second heart field. Here we provide the first analysis of the expression pattern of Xenopus islet-1 (Xisl-1) in the context of cardiovascular development. During early stages of heart development Xisl-1 is co-expressed with Nkx2.5 in the cardiac crescent in Xenopus supporting the notion of an initially single heart field. At subsequent stages of cardiogenesis the expression domains of Xisl-1 and Nkx2.5 become more distinct with Xisl-1 being detected more anterior to Nkx2.5, however both factors continue to be co-expressed in the dorsal mesocardium and pericardial roof of the linear heart tube. The presence of a cardiac Xisl-1 progenitor pool in an amphibian whose heart lacks an anatomically separated right ventricle is intriguing. Functional analyses show that Xisl-1 is required for normal heart development. Inhibition of Xisl-1 results in defects in heart morphogenesis and in the downregulation of early cardiac markers implicating a role for Xisl-1 in cardiac specification. Additionally, Xisl-1 loss-of-function affects the expression of several vascular markers demonstrating the involvement of Xisl-1 in vasculogenesis.

A molecular signature for the "master" heart cell.

The vertebrate heart comprises a variety of cell types, the majority of which are cardiomyocytes, smooth muscle and endothelial cells. Their origin is still an intriguing research topic and the question is whether these cells derive from a common or from multiple distinct progenitor cell(s). Three recent publications not only suggest the existence of a single progenitor cell that can give rise to cardiovascular lineages but additionally uncovered, at least in part, the molecular identity of such a multipotent precursor cell. These findings constitute major progress in the quest for stem-cell therapies for cardiac diseases.

What does it take to make a heart?

Ever increasing advances are being made in our quest to understand what it takes to direct pluripotent precursor cells to adopt a specific developmental fate. Eventually, the obvious goal is that targeted manipulation of these precursor cells will result in an efficient and reliable production of tissue-specific cells, which can be safely employed for therapeutic purposes. We have gained an incredible insight as to which molecular pathways are involved in governing neural, skeletal and cardiac muscle fate decisions. However, we still face the challenge of how to direct, for example, a cardiac fate in stem cells in the amounts needed to be employed for regenerative means. Equally importantly, we need to resolve critical questions such as: can the in vitro generated cardiomyocytes actually functionally replace damaged heart tissue? Here I will provide an overview of the molecules and signalling pathways that have first been demonstrated in embryological studies to function in cardiogenesis, and summarize how this knowledge is being applied to differentiate mouse and human embryonic stem cells into cardiomyocytes.

Non-canonical Wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells.

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6-10 days after co-culture, cardiac differentiation was determined by alpha-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7+/-0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3beta, we further determined whether the glycogen synthase kinase-3beta inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mM) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.

Xenopus flotillin1, a novel gene highly expressed in the dorsal nervous system.

The two paralogues of the Xenopus flotillin1 gene (flotillin1A and flotillin1B), which encodes a putative membrane-associated protein, were cloned from egg, cleavage, and tadpole cDNA libraries. Both mRNAs are present during oogenesis and cleavage stages. After the onset of zygotic transcription, flotillin1 transcripts are first expressed throughout the embryonic ectoderm and become enhanced in the presumptive neural ectoderm as the neural plate forms. As the neural tube forms and differentiates, flotillin1 transcripts become enriched in the dorsal half, with particularly high expression in dorsal primary neurons. At early tail bud stages, there is additional expression in the paraxial mesoderm. At late tail bud stages, flotillin1A is expressed in branchial arch mesenchyme, the overlying branchial ectoderm and in dorsal somitic mesoderm, whereas flotillin1B expression is more restricted in the dorsal neural tube and head sensory structures. This report is the first comprehensive developmental description in any animal of the expression pattern of this gene, whose protein product in several systems plays important roles in signal transduction events.

Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor.

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. We have used markers of pre-placodal ectoderm (PPE) (six1, eya1) to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.

Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos.

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.

Molecular asymmetries in the animal-vegetal axis of the Xenopus oocyte are well known to regulate the formation of gametes and germ layers. Likewise, many transplantation and explant studies demonstrate that maternal dorsalizing activities are localized to the future dorsal side of the embryo after fertilization, but to date only a few of the molecules involved in this process have been shown to be asymmetrically distributed. In this report, we identify two new aspects of the maternal regulation of dorsal-ventral fate asymmetry in Xenopus blastomeres: cytoplasmic polyadenylation of dorsal maternal mRNAs and localized Wnt8b signaling. Previous studies demonstrated that there are maternal, dorsal axis-inducing RNAs localized to dorsal animal blastomeres that become activated between the 8- and 16-cell stage (Hainski and Moody [1992] Development 116:347-355; Hainski and Moody [1996] Dev. Genet. 19:210-221). We report herein that the activation of these axis-inducing dorsal mRNAs is regulated by cytoplasmic polyadenylation. We also show that maternal wnt8b mRNA is concentrated in ventral animal blastomeres. These ventral cells and exogenous Wnt8b both inhibit the dorsal fate of neighboring blastomeres in culture, indicating that a maternal Wnt signal also contributes to segregating dorsal and ventral fates.

The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts.

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin.