Here we show that, in the mammalian heart, Irx3 and/or Irx5 directly inhibits the expression of Bmp10. In the Irx3;Irx5 DKO E12.5 hearts, Bmp10 is misexpressed in the endocardial cells that line the ventricular trabeculae and the endocardial cushions. The ectopic expression of Bmp10 in the endocardium supports our model of redundancy between Irx3 and Irx5 in repressing expression of this gene. By E14.5, however, Bmp10 returns to its myocardial-specific pattern, consistent with the restricted expression of Irx3 and Irx5 to myocardium at this stage. Bmp10 regulates cardiac morphogenesis. Bmp10-deficient mice do not survive past E10.5 due to a range of cardiovascular defects, including abnormal OFT and AV endocardial cushion development (Chen et al., 2004). Myocardial-specific Bmp10 overexpression leads to normal embryonic development but abnormal postnatal cardiomyocyte hypertrophic growth and subaortic narrowing (Chen et al., 2006). As proxy for the overexpression of Bmp10, we tested the effect of activating BMP signaling in the endocardium, and found a defect in cardiac ventricular septation, suggesting that ectopic endocardial expression of Bmp10 in the Irx3;Irx5 DKO contributes to the phenotype. This effect is specific, as we found that activation of BMP signaling within a tissue that is not known to be directly dependent on BMP signaling (in the DMP using Isl1::Cre) does not lead to defects in its formation.
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embryos produced a phenotype similar to that of nok mutants (Fig. 1N,O), we first characterized the role of this gene in cardiac morphogenesis. The nok gene encodes a membrane-associated guanylate kinase (MAGUK) with 68% identity to mammalian Pals1/Mpp5 and is required for the maintenance of epithelial cell polarity (Wei and Malicki, 2002; Horne-Badovinac et al., 2003). In mammalian cells, Pals1/Mpp5 functions as a scaffolding protein that links the transmembrane receptor Crumbs with Par6- PRKC (Hurd et al., 2003; Wang et al., 2004). We tested whether Nok is a functional homolog of mammalian Pals1/Mpp5 by injecting murine Pals1/MPP5 mRNA into nok mutants and morphants. Murine Pals1/MPP5 mRNA largely prevented ventral curvature of morphants (n=29/30 morphants rescued; Fig. 3A-C). To further evaluate the rescue efficiency of Pals1/MPP5 mRNA, we assayed the organization of the retinal pigmented epithelium (RPE), which is almost completely lost in nok mutants but was significantly recovered upon injection of murine Pals1/MPP5 mRNA (Fig. 3D-F). Finally, we assessed the recovery of heart function by quantifying the presence of circulation within peripheral blood vessels in nok morphants at 36 hpf. Injection of Pals1/MPP5 mRNA caused a significant increase in the number of nok/mpp5 morphants with functional circulation (Fig. 3G). The failure to completely rescue the nok morphant phenotype may be attributed to sequence variations between Nok and murine Pals1. Therefore, Nok is a functional homolog of murine Pals1/Mpp5.
Cardiac development is a spatiotemporally regulated multistep morphogenetic process that depends on the addition of progenitor cells from four different sources, including cells from the first heart field and second heart field (FHF and SHF), cells derived from cardiac neural crest cells (CNCCs) and cells derived from the pro-epicardial organ (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001; Verzi et al., 2005; Vincent and Buckingham, 2010). The SHF progenitor cells migrate to the pre-existing scaffold of the linear heart tube and contribute to the right ventricle, outflow tract (OFT) myocardium and to some endocardium at embryonic day (E) 8.5-10.25 (Kelly and Buckingham, 2002; Buckingham et al., 2005; Verzi et al., 2005; Ward et al., 2005). Perturbation of SHF deployment and progenitor differentiation leads to a spectrum of CHDs (Kelly, 2012) and is responsible for the majority of CHDs (Buckingham et al., 2005; Bruneau, 2008). The posterior SHF contributes to the dorsal mesenchymal protrusion (DMP), an essential structure for chamber septation (Snarr et al., 2007a). Abnormal differentiation and development of the posterior SHF has been associated with cardiac morphogenesis defects, such as atrial septal defect (ASD) and atrioventricular septal defect (AVSD) (Briggs et al., 2012). However, the molecules and the mechanisms that regulate posterior SHF development are not entirely clear.
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clarifying the role of PcG genes in heart development. The cardiac precursors are generated in the anterior lateral plate mesoderm and migrate ventromedially to form the linear heart tube, which possesses a highly regionalized structure (13). Complex remodeling of the heart tube and immigration of the neural crest cells occur during looping to complete morphogenesis of the heart. The genetic pathways regulating regionalization and these complex processes were recently identified (13). The cardiac homeobox gene Nkx2.5/Csx (Nkx2.5), a mammalian homologue of the Drosophila tinman (14, 15), is presumed to be a cardiac selector gene essential for either cardiogenesis or cardiac morphogenesis (16). This hypothesis is based on the fact that Nkx2.5-deficient embryos display defects in looping morphogenesis of the heart and cardiac gene expression of Hand1, atrial natriuretic factor (ANF), and myosin light chain 2v (MLC2v) (17–19), and that exogenous expression of dominant negative Nkx2.5 into early Xenopus blastomeres abrogates heart formation (20, 21). Several mutations in NKX2.5/CSX1 (NKX2.5) have been reported in patients with a variety of cardiac anomalies, including atrioven- tricular (AV) conduction delays, atrial septal defects, TOF, and DORV (22–24), further underscoring the importance of Nkx2.5 in cardiac development.
Lineage analyses in different vertebrate models have suggested that myocardial and endocardial progenitor cell populations have distinct developmental origins. In zebrafish, fate-mapping studies have shown that the entire myocardial and endocardial progenitor populations can be traced back to blastula stages (Keegan et al., 2004; Lee et al., 1994). After gastrulation, progenitor cells with endothelial and hematopoietic potential become positioned within the anterior lateral plate mesoderm (ALPM), which is located rostrally to a spatially separate population of heart and neural crest derivatives expressed 2 (hand2)-expressing myocardial progenitors. At this stage, endocardial progenitors express the endothelial marker genes tyrosine kinase with immunoglobulin-like and EGF- like domains 2 (tie2; also known as tek), T-cell acute lymphocytic leukemia 1 (tal1), and ets variant 2 (etv2; also known as etsrp) (Bussmann et al., 2007; Schoenebeck et al., 2007), supporting a common origin for endocardial and other endothelial cells. Lineage tracing of cells within the ALPM has shown that some cells contribute to the endocardium of the ventricular chamber (Schoenebeck et al., 2007). Similar studies in chick embryos showed that an endocardial progenitor cell population that is separate from myocardial progenitor cells arises at, or prior to, the primitive streak stage (Cohen-Gould and Mikawa, 1996; Lough and Sugi, 2000; Milgrom-Hoffman et al., 2011; Wei and Mikawa, 2000). These observations have contributed to a view (known as the ‘ pre-specification ’ model) that endocardial progenitors arise from a distinct lineage within the pre-cardiac mesoderm that is separate to that giving rise to myocardial cells.
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mRNA was less abundant, except in the myocardium of the AV canal region. In the heart at E9.5, cardiac jelly is rich in HA (Figure 1f) and versican (Figure 1g). By E10.5, endothelial cells in the AV canal and outflow tract transform into mesenchymal cells and invade the underlying matrix. The migrating cells express high levels of Has2 mRNA (Figure 1h) and stain strongly for extracellular HA (Figure 1f, inset). Later, Has2 mRNA is expressed by mesenchymal cells during ele- vation of the secondary palate and by hypertrophic chondrocytes within epiphysial growth plates (data not shown); this is consistent with a role for Has2- dependent HA synthesis in the expansion of soft tis- sues and in chondrogenesis (24). The relatively high level of expression of Has2 mRNA and its presence at sites of HA accumulation suggest that Has2 is a major source of HA during organogenesis.
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The atrioventricular canal (AVC) physically separates the atrial and ventricular chambers of the heart and plays a crucial role in the development of the valves and septa. Defects in AVC development result in aberrant heart morphogenesis and are a significant cause of congenital heart malformations. We have used a forward genetic screen in zebrafish to identify novel regulators of cardiac morphogenesis. We isolated a mutant, named wickham ( wkm ), that was indistinguishable from siblings at the linear heart tube stage but exhibited a specific loss of cardiac looping at later developmental stages. Positional cloning revealed that the wkm locus encodes transmembrane protein 2 (Tmem2), a single-pass transmembrane protein of previously unknown function. Expression analysis demonstrated myocardial and endocardial expression of tmem2 in zebrafish and conserved expression in the endocardium of mouse embryos. Detailed phenotypic analysis of the wkm mutant identified an expansion of expression of known myocardial and endocardial AVC markers, including bmp4 and has2 . By contrast, a reduction in the expression of spp1 , a marker of the maturing valvular primordia, was observed, suggesting that an expansion of immature AVC is detrimental to later valve maturation. Finally, we show that immature AVC expansion in wkm mutants is rescued by depleting Bmp4, indicating that Tmem2 restricts bmp4 expression to delimit the AVC primordium during cardiac development.
myocardium to balloon from the tube, OC CMs must break down their actin cytoskeleton to allow cell growth and elongation, a process that is dependent upon fine-tuning of Add3 function. We hypothesize that miR-143 acts throughout the heart tube to limit the production of new, intracellular Add3 protein, thus providing one level of Add3 regulation. Concomitantly, we speculate that other regionalized signals, such as a kinase (Fukata et al., 1999), might act to post-translationally promote the dissociation of Add3 from F-actin, thereby allowing the cytoskeleton to be redistributed throughout the cell. In the absence of miR-143 function, continued Add3 production might overwhelm other modes of post- translational regulation, leading to sustained Add3-actin complex formation and cytoskeletal polymerization. Discerning whether miR-143 acts in concert with other regionalized factors to influence cell morphology will be vital to appreciating the full role of the miR-143-add3 pathway in cardiac morphogenesis and, possibly, disease.
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and in the adult heart. Here we used a spatiotemporally regulated gene inactivation approach to delineate the role of Fog2 both in embryo and adult heart. We found that early myocardial expres- sion of Fog2 is required for cardiac morphogenesis and coronary vascular development. Later myocardial inactivation of Fog2 was compatible with normal heart development but resulted in heart failure associated with underdevelopment of coronary vessels. This was recapitulated by induced cardiomyocyte-restricted Fog2 inacti- vation in the adult heart, demonstrating a continuing requirement for Fog2 in maintenance of heart function and coronary vascula- ture. Mice that expressed only the mutant GATA4[V217G] protein in the myocardium also developed heart failure and dropout of coronary vasculature. Collectively, our results demonstrate that cardiomyocyte expression of Fog2 is required for maintenance of myocardial and coronary vessel function in the adult heart. Results
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The main purpose of the work reported here was to re examine the behavior of the TCA cycle enzymes during the development of B. emersonii in synchronous cultures. It was hoped that such a study might offer clues to the biochemical mechanisms underlying morphogenesis in this mold. In some respects, our results contrast with previous v/ork and the assumptions derived therefrom (Cantino, 19ol and 1966). Before considering the results of the
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along the dorso-ventral axis. Second, the cells of the lateral epidermis elongate coordinately starting from the most dorsal and proceeding towards the most ventral ones in a morphogenetic wave. Coupled with this dorsalward movement, the anterior/poste- rior (A/P) axis stretches along the dorsal edge. These two move- ments result in the dorsalmost cells of the epidermis forming two straight lines (Martinez Arias, 1993; Ring and Martinez Arias, 1993). Finally, the epidermal layers meet at the dorsal midline, the dorsalmost cells lose their polarity, cell stretching stops and the epithelial sheet seals the gap to form a continuous epidermis. We could say, by analogy, that epidermal morphogenesis reaches "entelechia" and epidermal cells initiate their final differentiation on completion of dorsal closure.
During osteogenesis, mesenchymal cells differentiate in oste- oblast lineage and produce a mineralized ECM that takes control of morphogenic events (Morris-Kay and Tucket, 1991). The ECM complex is formed by proteoglycans (PG), glycosaminoglycans (GAG), fibronectin, collagens, and other glycoproteins, which are differently distributed and organised in tissues and stages of development. It has been suggested a model of “dynamic reciproc- ity” between the ECM and cellular components such as the cytoskeleton (Giancotti and Ruoslahti, 1999). The ECM-cell- receptor-link transmits signals across the cell membrane in the cytoplasm, thereby initiating a cascade of events that culminates in the expression of specific genes (Adams and Watt, 1993). Much experimental evidence demonstrates that osteogenetic processes fall largely under the balanced control of interactions between cells and the ECM (Martins-Green and Bissel, 1995). Fibronectin, for example, is distributed in areas of skeletogenesis and controls the early stages of bone formation regulating the recruitment and commitment of osteoblasts to terminal differentiation (Gronowicz et al., 1991). Osteoblast-fibronectin interactions are required for bone morphogenesis, since fibronectin regulates the matrix as- sembly and interacts with type I collagen fibrils (Nordahl et al., 1995, Moursi et al., 1996). Collagen is another ECM molecule that provides a morphogenetic signal. It realizes a matrix-mediated tissue interaction fundamental for embryo chondrocranial morpho- genesis (Wood et al., 1991). Laminin, fibronectin, collagen type I Fig. 1. Increased expression of IL-6 mRNA in Crouzon and Apert
IMPORTANCE Septins are conserved filament-forming GTPases involved in a wide range of cellular events, such as cytokinesis, exocytosis, and morphogenesis. In Candida albicans, the most prevalent human fungal pathogen, septin functions are indis- pensable for its virulence. However, the molecular mechanisms by which septin structures are regulated are poorly understood. In this study, we deleted NAP1, a gene encoding a putative septin regulator, in C. albicans and found that cells lacking NAP1 showed abnormalities in morphology, invasive growth, and septin ring dynamics. We identified a conserved N-terminal phos- phorylation cluster on Nap1 and demonstrated that phosphorylation at these sites regulates Nap1 localization and function. Im- portantly, deletion of NAP1 or mutation in the N-terminal phosphorylation cluster strongly reduced the virulence of C. albicans in a mouse model of systemic infection. Thus, this study not only provides mechanistic insights into septin regulation but also suggests Nap1 as a potential antifungal target.
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Multiciliated cells (MCCs) possess multiple motile cilia and are distributed throughout the vertebrate body, performing important physiological functions by regulating fluid movement in the intercellular space. Neither their function during organ development nor the molecular mechanisms underlying multiciliogenesis are well understood. Although dysregulation of members of the miR-34 family plays a key role in the progression of various cancers, the physiological function of miR-34b, especially in regulating organ formation, is largely unknown. Here, we demonstrate that miR-34b expression is enriched in kidney MCCs and the olfactory placode in zebrafish. Inhibiting miR-34b function using morpholino antisense oligonucleotides disrupted kidney proximal tubule convolution and the proper distribution of distal transporting cells and MCCs. Microarray analysis of gene expression, cilia immunostaining and a fluid flow assay revealed that miR-34b is functionally required for the multiciliogenesis of MCCs in the kidney and olfactory placode. We hypothesize that miR-34b regulates kidney morphogenesis by controlling the movement and distribution of kidney MCCs and fluid flow. We found that cmyb was genetically downstream of miR-34b and acted as a key regulator of multiciliogenesis. Elevated expression of cmyb blocked membrane docking of centrioles, whereas loss of cmyb impaired centriole multiplication, both of which resulted in defects in the formation of ciliary bundles. Thus, miR-34b serves as a guardian to maintain the proper level of cmyb expression. In summary, our studies have uncovered an essential role for miR-34b-Cmyb signaling during multiciliogenesis and kidney morphogenesis.
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Studies comparing DNA methylation status in newborns with aortic valve stenosis (AVS), TOF, and VSDs have identified several aberrant patterns in diseased patients [75–77]. A total of 52 genes with significantly altered DNA methylation were identified in the blood of newborn AVS patients compared to newborn healthy infants. Of particular interest are APOA5 (a determinant of plasma triglyceride levels) and PCSK9 (an activator of low-density lipoprotein receptors), which were both hypermethylated and are both considered major risk factors for coronary artery disease in adults . In myocardial biopsies from patients with TOF and VSD, hypermethylation was observed in the promoter region of SCO2, which is a cytochrome c oxidase protein. The authors hypothesized that the resultant SCO2 downregulation drives the metabolic state of cardiac cells toward glycolysis, delaying terminal differentiation and promoting cardiomyopathy and heart failure . In another study assessing the DNA methylation of key cardiac transcription factors, Nkx2.5 and chamber-specific Hand1 were found to be hypermethylated in patients with TOF . As expected, the expression of these two transcription factors was found to be downregulated. In a revealing study, monozygotic twins discordant for DORV were analyzed for genomic and epigenomic differences . Not surprisingly, very few genetic differences were identified. However, 121 transcription factor binding sites were differentially methylated, including the hypermethylation of ZIC3 (a zinc finger protein involved in Wnt signaling) and NR2F2 (a protein important in heart development), in the diseased twin. This finding highlights the importance of epigenetics in diseases with complex etiologies, such as CHD.
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polymerization (Goode and Eck, 2007). Eukaryotic species have multiple formin proteins, including Dia, Fmn, Fmnl and Daam, the activities of which are regulated by diverse signals (Schonichen and Geyer, 2010). However, our current understanding of their physiological functions is quite limited, particularly in mammals. Through their actin regulating capacity, formins probably impact tissue morphogenesis, a biological/developmental process involving complex signaling pathways mediated by cytoskeletal dynamics (Zallen, 2007). Indeed, Fmn1 was found to be crucial for limb development in mouse (Zhou et al., 2009). Surprisingly, Dia1 (Diap1 – Mouse Genome Informatics) knockout mice were developmentally/morphologically normal, albeit with an age- dependant myeloproliferative defect (Peng et al., 2007). Despite that, Dia1 displays significantly stronger activity in promoting actin nucleation than do other formin family members (Higashi et al., 2008). These findings strongly suggest that each formin plays a unique and specific physiological function, largely depending on its individual expression profile, upstream activators, localization and inherent activity.
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Although the results of this study has suggested the functional importance of TACE during lung growth and development, the regulation of expression of ADAM family members including TACE is poorly understood. Physiological stimuli such as acetylcholine and lipopolysaccharide, or synthetic compounds, such as phorbol esters, dramatically increase the release of numerous shed pro- teins (Hooper et al., 1997). To elucidate the general mechanisms that regulate ectodomain shedding, TGF- α cleavage is found to be induced by fibroblast growth factor or platelet-derived growth factors and is critically dependent on the ERK/MAP kinase path- way (Fan and Derynck, 1999). On the other hand, TIMP-3, but not TIMP-1, 2, or 4, is a potent inhibitor of TACE activity (Amour et al., 1998). Since we have shown herein that TACE-mediated shedding plays a functional role during lung branching morphogenesis and cytodifferentiation, interpreting the regulatory mechanism of TACE catalytic activity may further our understanding of the biological significance of TACE in the developing lung tissue.
Dental morphogenesis is a complex multi-factorial process where differential mitotic activities, apoptosis as well as cell migra- tion and cell adhesion may play an important role (Gaunt, 1955, 1956; Cohn, 1957; Butler and Ramadan, 1962; Ruch, 1984; Palacios et al., 1995; Lesot et al., 1996; Tureckova et al., 1996; Vaahtokari et al., 1996a; Fausser et al., 1998; Peterková et al., 1998). The number and the spatial distribution of the functional post-mitotic cells (odontoblasts, ameloblasts), specific for each type of tooth, determine the final shape of the crown. The functional mouse incisor shows a single cusp and enamel is present only on its labial side. The absence of enamel on its lingual surface has been suggested to result from a lack of competence of cells of the lingual inner dental epithelium (Amar et al., 1987). The continuously growing rodent incisor is a widely used model to investigate
As ihha is expressed all along the vp edge, yet only appears to control the morphology of the most anterior region, we examined whether its co-ortholog, ihhb, may function redundantly with ihha. Expression of ihhb is not apparent in any bone-forming cells associated with the Op of either wild-type or ihha mutant larvae (supplementary material Fig. S4). However, we note that ihhb expression is visible in a domain just dorsal to the jp edge of the Op that corresponds to the location of muscles that elevate the Op (supplementary material Fig. S4). Thus, Ihhb could possibly signal to the posterior portion of the vp edge and compensate locally for the loss of ihha. Therefore, we used cyclopamine (CyA) to inhibit all Hh signaling from 4-6 dpf and test whether other Hh ligands than Ihha contribute to Op morphogenesis. We find that addition of CyA either to wild-type or ihha mutant larvae yields a quantitatively equivalent Op phenotype to untreated ihha mutants (Fig. 8). That CyA treatment matches and does not exacerbate the ihha mutant phenotype suggests that Ihha is the only Hh ligand required for early Op patterning and, furthermore, that the requirement for Hh signaling is during the 4-6 dpf time window, corresponding to the period of early Op morphogenesis when we first observe the ihha mutant phenotype.
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The role of RA in pharyngeal patterning in the mouse (see Introduction) is restricted to the 7-10 somite period (Wendling et al., 2000), corresponding approximately to E8.5. The expression do- mains of some Nkx2 genes became restricted in the anterior- posterior and medio-lateral axes of the pharynx at or even before this time. Three genes from of cardiac Nkx2 clade (Nkx2-3, Nkx2- 5 and Nkx2-6) are expressed in the mouse pharynx (Biben et al., 1998; Lints et al., 1993; Pabst et al., 1997). Initially, all are expressed in the full medio-lateral extent of the pharyngeal floor (Fig. 2 E-G, L; Biben et al., 1998; Stanley et al., 2002). However, Nkx2-6 becomes restricted around E8.0 to the lateral regions encompassing the pharyngeal pouches (Fig. 2B), and thus be- comes relatively downregulated in midline precursors of the lungs, thyroid, salivary glands and oral cavity (Biben et al., 1998). Con- versely, around the same time, Nkx2-5 expression withdraws from Fig. 1. RNase protection analysis of Nkx2-3 expression. The panel shows Nkx2-3 and control cyclophilin transcripts (arrowheads) detected by RNase protection in samples of total RNA extracted from adult tissues. Yeast tRNA was included as negative control.