Regulation of cardiogenesis by putative WNT signalling pathways

PhD ThesisThe Wnt/ -catenin and the Wnt/planar cell polarity (Wnt/PCP) signalling pathways have been shown to play important roles in cardiogenesis and their disruption has been shown to cause severe disturbances in heart development. Spatially and temporally complex interplays between the two pathways have been described. One component of the PCP pathway is Jnk, a member of the highly conserved mitogenactivated protein kinase (MAPK) family. This stress responsive mitogen is known to control a variety of cellular behaviours such as proliferation, apoptosis and cell migratory behaviour and as such, is likely to be of pivotal importance in cardiac development. The aim of this study was to investigate the role played by Jnk in vertebrate heart formation and the relationships between Jnk signalling and canonical Wnt signalling, using in silico and in vivo approaches in zebrafish and an in vitro approach on a mouse embryonic stem (ES) cell model of cardiogenesis. Firstly, using a range of bioinformatic methods, an analysis of jnk genes, splice variants and proteins, and an investigation of their phylogenetic relation with other species was undertaken. This suggested conservation of Jnk family members, but suggested that there were additional orthologues of jnk1 present in the zebrafish transcriptome. The spatial and temporal expression profiles of these genes were then examined by semi-quantitative PCR and in situ hybridisation. The functional role of Jnk proteins during zebrafish development was subsequently investigated using a specific chemical inhibitor, SP600125. Inhibition of Jnk signalling during gastrulation and somitogenesis caused a convergence extension-like phenotype and severe cardiac defects, including looping anomalies and alterations in atrial versus ventricular cell numbers. ES cells have the capacity to differentiate in vitro and give rise to cells of many different lineages, including cardiomyocytes. Canonical Wnt and Jnk components were manipulated during specific windows of differentiation as ES cells formed beating embryoid bodies. Examination of the spontaneous contractile behaviour of differentiating ES cells as they entered the cardiogenic lineage, and analysis of their developmental gene expression profiles, showed the beating behaviour of ES cellderived cardiac cells was enhanced in a temporally specific manner after inhibition of the non-canonical Wnt/Jnk pathway, while there was marked alteration of canonical Wnt signalling. To investigate whether there were reciprocal interactions between the two pathways, analysis of the system after activation of the canonical pathway was also undertaken. These studies indicated that the beating behaviour of ES cell-derived cardiac cells was enhanced in a temporally specific manner after inhibition of Jnk, while after activation of canonical Wnt/ -catenin signalling, the cardiogenic potential of differentiating ES cells was severely suppressed. The findings of this study extend our understanding of the role played by canonical and non-canonical Wnt signalling pathways in heart morphogenesis and highlight the interacting effects of related signalling pathways activity in cardiogenesis

Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia

Bmp2 and Notch cooperate to pattern the embryonic endocardium

International audienceSignaling interactions between the myocardium and endocardium pattern embryonic cardiac regions, instructing their development to fulfill specific functions in the mature heart. We show that ectopic Bmp2 expression in the mouse chamber myocardium changes the transcriptional signature of adjacent chamber endocardial cells into valve tissue, and enables them to undergo epithelial-mesenchyme transition. This induction is independent of valve myocardium specification and requires high levels of Notch1 activity. Biochemical experiments suggest thatBmp2-mediated Notch1 induction is achieved through transcriptional activation of the Notch ligand Jag1, and physical interaction of Smad1/5 with the intracellular domain of the Notch1 receptor. Thus, widespread myocardial Bmp2 and endocardial Notch signaling drive presumptive ventricular endocardium to differentiate into valve endocardium. Understanding the molecular basis of valve development is instrumental to designing therapeutic strategies for congenital heart valve defects

Myocardial Bmp2 gain causes ectopic EMT and promotes cardiomyocyte proliferation and immaturity

International audienceDuring mammalian heart development, restricted myocardial Bmp2 expression is a key patterning signal for atrioventricular canal specification and the epithelial-mesenchyme transition that gives rise to the valves. Using a mouse transgenic line conditionally expressing Bmp2, we show that widespread Bmp2 expression in the myocardium leads to valve and chamber dysmorphogenesis and embryonic death by E15.5. Transgenic embryos show thickened valves, ventricular septal defect, enlarged trabeculae and dilated ventricles, with an endocardium able to undergo EMT both in vivo and in vitro. Gene profiling and marker analysis indicate that cellular proliferation is increased in transgenic embryos, whereas chamber maturation and patterning are impaired. Similarly, forced Bmp2 expression stimulates proliferation and blocks cardiomyocyte differentiation of embryoid bodies. These data show that widespread myocardial Bmp2 expression directs ectopic valve primordium formation and maintains ventricular myocardium and cardiac progenitors in a primitive, proliferative state, identifying the potential of Bmp2 in the expansion of immature cardiomyocytes

Msx1 cre ERT 2 knock-In allele: A useful tool to target embryonic and adult cardiac valves

Heart valve development begins with the endothelial-to-mesenchymal transition (EMT) of endo-cardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate-mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock-in tamoxifen-inducible Cre line was recently generated (Msx1 CreERT2) and characterized during embryonic development and after birth, and was shown to recapit-ulate the endogenous Msx1 expression pattern. Here, we further analyze this knock-in allele and track the Msx1-expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1 CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling

Alternative splicing of jnk1a in zebrafish determines first heart field ventricular cardiomyocyte numbers through modulation of hand2 expression.

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia

Abnormalities in formation of the craniofacial bones in <i>RockDN;Wnt1-cre</i> embryos.

<p><b>A,B</b>) In severely affected <i>RockDN;Wnt1-cre</i> embryos at E14.5, the frontonasal bones (stained with alcian blue) are absent (arrow in B, compare to A). Meckel's cartilage is also reduced in size (arrowhead in B, compare with A). <b>C,D</b>) Bone (red) and cartilage (blue) staining of a mildly affected <i>RockDN;Wnt1-cre</i> embryo at E18.5 (D), shows that the maxilla (arrowhead) and mandibular (arrow) bones are well formed, although the hyoid bone (red arrow) is reduced in size in mutant embryos. <b>E,F</b>) Inferior views of the base of the skull in mildly affected embryos shows that the basisphenoid and the presphenoid bones are hypoplastic in <i>RockDN;Wnt1-cre</i> embryos, whereas the nasal septum is completely missing. Moreover, the maxillary bones are widely separated in mutant embryos (double arrow in F), compared to control littermates (E). bs = basisphenoid; n = nasal septum; ps = presphenoid. Scale bar = 500 µm.</p

Heterotopic ossification in mice overexpressing Bmp2 in Tie2+ lineages.

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation

Ectopic and excessive cell death in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A–D are sections through the neural tube (line i in M), E–H through the pharyngeal arch 1 (line i in M) and I–L through the frontonasal processes (line ii in M). A,C,E,G,I,K show caspase 3-expressing cells (red) and Wnt1-cre+ve NCCs (green). B,D,F,H,J,L are the same sections but only showing the caspase 3-expressing cells. <b>A–D</b>) Whereas only very occasional activated caspase 3-expressing, dying, cells (red) are seen in the neural epithelium in control embryos, there are many dying cells observed in the dorsal part of the neural tube, from which the NCC emerge, in <i>RockDN;Wnt1-cre</i> mutant embryos (arrows in C and D). <b>E–L</b>) Very few activated caspase3-expressing cells are observed in the NCC-derived ectomesenchyme of pharyngeal arch 1 (E,F) and the frontonasal region (I,J) in control embryos. In contrast, many dying cells are seen in corresponding regions from <i>RockDN;Wnt1-cre</i> mutants (G,H,K,L). The surface ectoderm in the mutant is more irregular, compared to controls (arrowheads in E,G,I,K) and the inner NCC-derived ectomesenchyme is loosely arranged with gaps between the cells (arrows in G,K). <b>N,O</b>) The mean apoptotic and mitotic indexes were calculated for NCC within E9.5 pharyngeal arches. There is a significant increase in cell death in the mutant samples compared to controls (P = 0.019; * in N). There is no significant difference in cell proliferation between the two samples (P = 0.433; O). Scale bar = 50 µm.</p

Disruption of the actin cytoskeleton and vinculin-containing focal contacts in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A,B,E,F,I,J,M,N (line i in Q) are sections from first pharyngeal arch and C,D,G,H,K,L,O,P (line ii in Q) are from the frontonasal processes. A–H show phalloidin (red) and caspase 3 (green) immunofluorescence, with E–H being magnified regions as shown by the boxes in A–D, respectively. I–L show vinculin (red) and caspase 3 (green) dual immunofluorescence, with M–P being magnified regions as shown by boxes in I–L. The dotted lines in C,D,G,H,K,L,O,P indicate the boundary between the inner ectomesenchyme and the neural ectoderm and the surface ectoderm. <b>A–H</b>) Filamentous actin, labelled with phalloidin (red) outlines the cells in NCC-derived ectomesenchyme and neural ectoderm in the first pharyngeal arch (A,E) and frontonasal processes (C,G) of control embryos. Cortical phalloidin staining is lost in the ectomesenchyme from <i>RockDN;Wnt1-cre</i> mutants (F,H) but is maintained in the neural ectoderm (compare G with H). In addition, intense phalloidin-labelled foci are observed throughout the ectomesenchyme of the <i>RockDN;Wnt1-cre</i> mutants (dense red foci, blue arrow in F,H). Green caspase 3-positive cells are interspersed (white arrow) and overlapping with the phalloidin-intense cells (arrowheads in F,H). <b>I–L</b>) Vinculin and caspase 3 staining. In the pharyngeal arch the vinculin staining is not restricted to the centre of the arch in the mutant (compare J with I). In the frontonasal processes vinculin outlines the boundary between the surface ectoderm and the neural ectoderm with the inner NCC-derived ectomesenchyme (dotted lines in K,L). This discrete vinculin staining is lost in the <i>RockDN;Wnt1-cre</i> mutants (compare P with O). cas3 = activated caspase-3; mes = mesenchyme; ne = neural ectoderm; phall = phalloidin; se = surface ectoderm; vin = vinculin. Scale bar = 50 µm.</p