Notch and Hedgehog in the thymus/parathyroid common primordium: Crosstalk in organ formation.

The avian thymus and parathyroids (T/PT) common primordium derives from the endoderm of the third and fourth pharyngeal pouches (3/4PP). The molecular mechanisms that govern T/PT development are not fully understood. Here we study the effects of Notch and Hedgehog (Hh) signalling modulation during common primordium development using in vitro, in vivo and in ovo approaches. The impairment of Notch activity reduced Foxn1/thymus-fated and Gcm2/Pth/parathyroid-fated domains in the 3/4PP and further compromised the development of the parathyroid glands. When Hh signalling was abolished, we observed a reduction in the Gata3/Gcm2- and Lfng-expression domains at the median/anterior and median/posterior territories of the pouches, respectively. In contrast, the Foxn1 expression-domain at the dorsal tip of the pouches expanded ventrally into the Lfng-expression domain. This study offers novel evidence on the role of Notch signalling in T/PT common primordium development, in an Hh-dependent manner.

The notch ligand delta-like 4 regulates multiple stages of early hemato-vascular development.

BACKGROUND: In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4(+/-) and Dll4(-/-) embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis. CONCLUSIONS/SIGNIFICANCE: This study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis.

Portrayal of the Notch system in embryonic stem cell-derived embryoid bodies.

We portrayed the Notch system in embryonic stem cell (ESC)-derived embryoid bodies (EBs) differentiating under the standard protocols used to assess yolk sac (YS) hematopoiesis in vitro. Notch receptors and Notch ligands were detected in virtually all cells throughout EB development. Notch 1 and Notch 2, but not Notch 4, were visualized in the nucleus of EB cells, and all these receptors were also observed as patent cytoplasmic foci. Notch ligands (Delta-like 1 and 4, Jagged 1 and 2) were immunodetected mostly as cytoplasmic foci. Widespread Notch 1 activation was evident at days 2-4 of EB differentiation, the time window of hemangioblast generation in this in vitro system. EBs experienced major spatial remodeling beyond culture day 4, the time point coincident with the transition between primitive and multilineage waves of YS hematopoiesis in vitro. At day 6, where definitive YS hematopoiesis is established in EBs, these exhibit an immature densely packed cellular region (DCR) surrounded by a territory of mesodermal-like cells and an outer layer of endodermal cells. Immunolabeling of Notch receptors and ligands was usually higher in the DCR. Our results show that Notch system components are continuously and abundantly expressed in the multicellular environments arising in differentiating EBs. In such an active Notch system, receptors and ligands do not accumulate extensively at the cell surface but instead localize at cytoplasmic foci, an observation that fits current knowledge on endocytic modulation of Notch signaling. Our data thus suggest that Notch may function as a territorial modulator during early development, where it may eventually influence YS hematopoiesis.

Hyalinizing clear cell carcinoma of salivary glands: a study of extracellular matrix.

Hyalinizing clear cell carcinoma (HCCC) is a recently described low-grade carcinoma of the salivary glands presenting two main histological features: clear neoplastic cells and prominent hyalinized stroma. Our aim was to characterize the composition of the hyalinized stroma in three cases of HCCC. An immuno-histochemical study using laminin and type I, III and IV collagens, fibronectin and tenascin antibodies was performed. In one case, electron microscopy study was also done. Collagen I and fibronectin were always present in tumoral stroma. Collagen III was documented in two cases and tenascin at the invasive front of the neoplasm. Collagen IV and laminin were found around neoplastic cells but not at the interstitial stroma. Foci of basal lamina reduplication and large deposits of long space collagen were observed in EM. This study demonstrates that the hyalinized stroma of HCCC is not formed by the accumulation of basement membrane-type material since it is mostly composed by collagen I and fibronectin.

Oocyte Ca2+ spike acquisition during in vitro development of early preantral follicles: influence of age and hormonal supplementation.

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.