Transcriptional responses to salinity-induced changes in cell wall morphology of the euryhaline diatom Pleurosira laevis.

Diatoms are unicellular algae with morphologically diverse silica cell walls, which are called frustules. The mechanism of frustule morphogenesis has attracted attention in biology and nanomaterials engineering. However, the genetic regulation of the morphology remains unclear. We therefore used transcriptome sequencing to search for genes involved in frustule morphology in the centric diatom Pleurosira laevis, which exhibits morphological plasticity between flat and domed valve faces in salinity 2 and 7, respectively. We observed differential expression of transposable elements (TEs) and transporters, likely due to osmotic response. Up-regulation of mechanosensitive ion channels and down-regulation of Ca2+-ATPases in cells with flat valves suggested that cytosolic Ca2+ levels were changed between the morphologies. Calcium signaling could be a mechanism for detecting osmotic pressure changes and triggering morphological shifts. We also observed an up-regulation of ARPC1 and annexin, involved in the regulation of actin filament dynamics known to affect frustule morphology, as well as the up-regulation of genes encoding frustule-related proteins such as BacSETs and frustulin. Taken together, we propose a model in which salinity-induced morphogenetic changes are driven by upstream responses, such as the regulation of cytosolic Ca2+ levels, and downstream responses, such as Ca2+-dependent regulation of actin dynamics and frustule-related proteins. This study highlights the sensitivity of euryhaline diatoms to environmental salinity and the role of active cellular processes in controlling gross valve morphology under different osmotic pressures.

Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis.

RATIONALE: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. OBJECTIVE: The aim of this study is to determine the functional specificity of Notch in valve development. METHODS AND RESULTS: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. CONCLUSIONS: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.

Notch signaling functions in lymphatic valve formation.

Collecting lymphatic ducts contain intraluminal valves that prevent backflow. In mice, lymphatic valve morphogenesis begins at embryonic day 15.5 (E15.5). In the mesentery, Prox1 expression is high in valve-forming lymphatic endothelial cells, whereas cells of the lymphatic ducts express lower levels of Prox1. Integrin α9, fibronectin EIIIA, Foxc2, calcineurin and the gap junction protein Cx37 are required for lymphatic valve formation. We show that Notch1 is expressed throughout the developing mesenteric lymphatic vessels at E16.5, and that, by E18.5, Notch1 expression becomes highly enriched in the lymphatic valve endothelial cells. Using a Notch reporter mouse, Notch activity was detected in lymphatic valves at E17.5 and E18.5. The role of Notch in lymphatic valve morphogenesis was studied using a conditional lymphatic endothelial cell driver either to delete Notch1 or to express a dominant-negative Mastermind-like (DNMAML) transgene. Deletion of Notch1 led to an expansion of Prox1(high) cells, a defect in Prox1(high) cell reorientation and a decrease in integrin α9 expression at sites of valve formation. Expression of DNMAML, which blocks all Notch signaling, resulted in a more severe phenotype characterized by a decrease in valves, failure of Prox1(high) cells to cluster, and rounding of the nuclei and decreased fibronectin-EIIIA expression in the Prox1(high) cells found at valve sites. In human dermal lymphatic endothelial cells, activation of Notch1 or Notch4 induced integrin α9, fibronectin EIIIA and Cx37 expression. We conclude that Notch signaling is required for proper lymphatic valve formation and regulates integrin α9 and fibronectin EIIIA expression during valve morphogenesis.

Dynamic Expression Profiles of ß-Catenin during Murine Cardiac Valve Development.

ß-catenin has been widely studied in many animal and organ systems across evolution, and gain or loss of function has been linked to a number of human diseases. Yet fundamental knowledge regarding its protein expression and localization remains poorly described. Thus, we sought to define whether there was a temporal and cell-specific regulation of ß-catenin activities that correlate with distinct cardiac morphological events. Our findings indicate that activated nuclear ß-catenin is primarily evident early in gestation. As development proceeds, nuclear ß-catenin is down-regulated and becomes restricted to the membrane in a subset of cardiac progenitor cells. After birth, little ß-catenin is detected in the heart. The co-expression of ß-catenin with its main transcriptional co-factor, Lef1, revealed that Lef1 and ß-catenin expression domains do not extensively overlap in the cardiac valves. These data indicate mutually exclusive roles for Lef1 and ß-catenin in most cardiac cell types during development. Additionally, these data indicate diverse functions for ß-catenin within the nucleus and membrane depending on cell type and gestational timing. Cardiovascular studies should take into careful consideration both nuclear and membrane ß-catenin functions and their potential contributions to cardiac development and disease.

Frustule morphogenesis of raphid pennate diatom Encyonema ventricosum (Agardh) Grunow.

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.