Endocardial to Myocardial Notch-Wnt-Bmp Axis Regulates Early Heart Valve Development

Endocardial to mesenchymal transformation (EMT) is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC) endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1) show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.     

Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2

The establishment of chamber specificity is an essential requirement for cardiac morphogenesis and function. Hesr1 (Hey1) and Hesr2 (Hey2) are specifically expressed in the atrium and ventricle, respectively, implicating these genes in chamber specification. In our current study, we show that the forced expression of Hesr1 or Hesr2 in the entire cardiac lineage of the mouse results in the reduction or loss of the atrioventricular (AV) canal. In the Hesr1-misexpressing heart, the boundaries of the AV canal are poorly defined, and the expression levels of specific markers of the AV myocardium, Bmp2 and Tbx2, are either very weak or undetectable. More potent effects were observed in Hesr2-misexpressing embryos, in which the AV canal appears to be absent entirely. These data suggest that Hesr1 and Hesr2 may prevent cells from expressing the AV canal-specific genes that lead to the precise formation of the AV boundary. Our findings suggest that Tbx2 expression might be directly suppressed by Hesr1 and Hesr2. Furthermore, we find that the expression of Hesr1 and Hesr2 is independent of Notch2 signaling. Taken together, our data demonstrate that Hesr1 and Hesr2 play crucial roles in AV boundary formation through the suppression of Tbx2.     

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

Notch activity is required to maintain floorplate identity and to control neurogenesis in the chick hindbrain and spinal cord

Notch signalling plays a major role in many invertebrate and vertebrate patterning systems. In this paper, we use high-titre, non-replicative pseudotype viruses to show that the two Notch ligands, Delta1 and Serrate1 (Jagged1), have differing activities in the developing chick spinal cord and hindbrain. In the walls of the neural tube, Serrate1 appears not to affect neurogenesis, in contrast to Delta1 which mediates lateral inhibition as elsewhere in the nervous system. In the floorplate we find that there is also a requirement for Notch, but with a different type of dependence on the two Notch ligands: cells with a floorplate character are lost when Notch activity is blocked with dominant-negative, truncated forms of either Delta1 or Serrate1. Our results are consistent with ligand-receptor specificity within the Notch signalling pathway, Serrate1 recognising selectively Notch2 (which is expressed in the floorplate), and Delta1 acting on both Notch2 and Notch1 (which is expressed in the walls of the neural tube).     

Notch1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis

Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722-725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as gamma-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation.     

Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.     

Complementation of human papillomavirus type 16 E6 and E7 by Jagged1-specific Notch1-phosphatidylinositol 3-kinase signaling involves pleiotropic oncogenic functions independent of CBF1;Su(H);Lag-1 activation

We have analyzed the induction and role of phosphatidylinositol 3-kinase (PI3K) by Notch signaling in human papillomavirus (HPV)-derived cancers. Jagged1, in contrast to Delta1, is preferentially upregulated in human cervical tumors. Jagged1 and not Delta1 expression sustained in vivo tumors by HPV16 oncogenes in HaCaT cells. Further, Jagged1 expression correlates with the rapid induction of PI3K-mediated epithelial-mesenchymal transition in both HaCaT cells and a human cervical tumor-derived cell line, suggestive of Delta1;Serrate/Jagged;Lag2 ligand-specific roles. Microarray analysis and dominant-negatives reveal that Notch-PI3K oncogenic functions can be independent of CBF1;Su(H);Lag-1 activation and instead relies on Deltex1, an alternative Notch effector.     

Wnt signaling regulates atrioventricular canal formation upstream of BMP and Tbx2

In the developing heart, the atrioventricular canal (AVC) is essential for separation and alignment of the cardiac chambers, for valve formation, and serves to delay the electrical impulse from the atria to the ventricles. Defects in various aspects of its formation are the most common form of congenital heart defects. Using mutant and transgenic approaches in zebrafish, this study demonstrates that Wnt/β-catenin signaling is both sufficient and required for the induction of BMP4 and Tbx2b expression in the AVC and consequently the proper patterning of the myocardium. Furthermore, genetic analysis shows that Wnt/β-catenin signaling is upstream and in a linear pathway with BMP and Tbx2 during AVC specification.     

Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear

Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, we have found that ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches. The Notch ligand Serrate1 (Jag1 in mouse and humans) is expressed in the patches from an early stage and may provide Notch activation during the prosensory phase. Here, we test whether Notch signalling is actually required for prosensory patch development. When we block Notch activation in the chick embryo using the gamma-secretase inhibitor DAPT, we see a complete loss of prosensory epithelial cells in the anterior otocyst, where they are diverted into a neuroblast fate via failure of Delta1-dependent lateral inhibition. The cells of the posterior prosensory patch remain epithelial, but expression of Sox2 and Bmp4 is drastically reduced. Expression of Serrate1 here is initially almost normal, but subsequently regresses. The patches of sensory hair cells that eventually develop are few and small. We suggest that, in normal development, factors other than Notch activity initiate Serrate1 expression. Serrate1, by activating Notch, then drives the expression of Sox2 and Bmp4, as well as expression of the Serrate1 gene itself. The positive feedback maintains Notch activation and thereby preserves and perhaps extends the prosensory state, leading eventually to the development of normal sensory patches.     

Manic fringe and lunatic fringe modify different sites of the Notch2 extracellular region, resulting in different signaling modulation

Three mammalian fringe proteins are implicated in controlling Notch activation by Delta/Serrate/Lag2 ligands during tissue boundary formation. It was proved recently that they are glycosyltransferases that initiate elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats in the extracellular domain of Notch molecules. Here we demonstrate the existence of functional diversity among the mammalian fringe proteins. Although both manic fringe (mFng) and lunatic fringe (lFng) decreased the binding of Jagged1 to Notch2 and not that of Delta1, the decrease by mFng was greater in degree than that by lFng. We also found that both fringe proteins reduced Jagged1-triggered Notch2 signaling, whereas neither affected Delta1-triggered Notch2 signaling. However, the decrease in Jagged1-triggered Notch2 signaling by mFng was again greater than that by lFng. Furthermore, we observed that each fringe protein acted on a different site of the extracellular region of Notch2. Taking these findings together, we propose that the difference in modulatory function of multiple fringe proteins may result from the distinct amino acid sequence specificity targeted by these glycosyltransferases.     

Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2

Proteins encoded by the fringe family of genes are required to modulate Notch signalling in a wide range of developmental contexts. Using a cell co-culture assay, we find that mammalian Lunatic fringe (Lfng) inhibits Jagged1-mediated signalling and potentiates Delta1-mediated signalling through Notch1. Lfng localizes to the Golgi, and Lfng-dependent modulation of Notch signalling requires both expression of Lfng in the Notch-responsive cell and the Notch extracellular domain. Lfng does not prevent binding of soluble Jagged1 or Delta1 to Notch1-expressing cells. Lfng potentiates both Jagged1- and Delta1-mediated signalling via Notch2, in contrast to its actions with Notch1. Our data suggest that Fringe-dependent differential modulation of the interaction of Delta/Serrate/Lag2 (DSL) ligands with their Notch receptors is likely to have a significant role in the combinatorial repertoire of Notch signalling in mammals.