The role of the Notch signalling pathway in regulating the balance between neuronal and nonneuronal cells in sympathetic ganglia and the adrenal gland.

Sympathetic neurons and endocrine chromaffin cells of the adrenal medulla are catecholaminergic cells that derive from the neural crest. According to the classic model, they develop from a common sympathoadrenal (SA) progenitor that has the ability to differentiate into both sympathetic neurons and chromaffin cells depending on signals provided by their final environment. Our previous data revealed that a single premigratory neural crest cell can give rise to both sympathetic neurons and chromaffin cells, indicating that the fate decision between these cell types occurs after delamination. A more recent study demonstrated that at least half of chromaffin cells arise from a later contribution by Schwann cell precursors. Since Notch signalling is known to be implicated in the regulation of cell fate decisions, we investigated the early role of Notch signalling in regulating the development of neuronal and non-neuronal SA cells within sympathetic ganglia and the adrenal gland. To this end, we implemented both gain and loss of function approaches. Electroporation of premigratory neural crest cells with plasmids encoding Notch inhibitors revealed an elevation in the number of SA cells expressing the catecholaminergic enzyme tyrosine-hydroxylase, with a concomitant reduction in the number of cells expressing the glial marker P0 in both sympathetic ganglia and adrenal gland. As expected, gain of Notch function had the opposite effect. Numbers of neuronal and non-neuronal SA cells were affected differently by Notch inhibition depending on the time of its onset. Together our data show that Notch signalling can regulate the ratio of glial cells, neuronal SA cells and nonneuronal SA cells in both sympathetic ganglia and the adrenal gland.

MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells.

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells.

Sympathetic neurons and chromaffin cells share a common progenitor in the neural crest in vivo.

BACKGROUND: The neural crest (NC) is a transient embryonic structure unique to vertebrates, which generates peripheral sensory and autonomic neurons, glia, neuroendocrine chromaffin and thyroid C-cells, melanocytes, and mesenchymal derivatives such as parts of the skull, heart, and meninges. The sympathoadrenal (SA) cell lineage is one major sub-lineage of the NC that gives rise to sympathetic neurons, chromaffin cells, and the intermediate small intensely fluorescent (SIF) cells. A key question is when during NC ontogeny do multipotent progenitors segregate into the different NC-derived lineages. Recent evidence suggested that sympathetic, sensory, and melanocyte progenitors delaminate from the thoracic neural tube (NT) in successive, largely non-overlapping waves and that at least certain NC progenitors are already fate-restricted within the NT. Whether sympathetic neurons and chromaffin cells, suggested by cell culture studies to share a common progenitor, are also fate segregated in ovo prior to emigration, is not known. RESULTS: We have conducted single cell electroporations of a GFP-encoding plasmid into the dorsal midline of E2 chick NTs at the adrenomedullary level of the NC. Analysis of their derivatives, performed at E6, revealed that in most cases, labelled progeny was detected in both sympathetic ganglia and adrenal glands, where cells co-expressed characteristic marker combinations. CONCLUSIONS: Our results show that sympathetic neurons and adrenal chromaffin cells share a common progenitor in the NT. Together with previous findings we suggest that phenotypic diversification of these sublineages is likely to occur after delamination from the NT and prior to target encounter.

The LIM-Homeodomain transcription factor Islet-1 is required for the development of sympathetic neurons and adrenal chromaffin cells.

Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2ß were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.

The Effect of an Aqueous Extract of Teucrium polium on Glutathione Homeostasis In Vitro: A Possible Mechanism of Its Hepatoprotectant Action.

Background. Teucrium polium is used in Arab traditional medicine to treat liver diseases. Glutathione is an important intracellular antioxidant, and intrahepatic glutathione levels are depleted in liver diseases. Hypothesis and Aim. This investigation tested the hypothesis that aqueous extracts of T. polium maintains intracellular glutathione levels by augmenting glutathione peroxidase and glutathione reductase activity in cultured hepatocytes. Methods. The effects of increasing concentrations (0.01-1 mg/mL) of aqueous extract of T. polium were assessed in cultured HepG2 cells following 24 hours incubation on (1) cellular integrity using (a) the Trypan blue exclusion assay, (b) the [di-methylthiazol-2yl]-2,5-diphenyl-tetrazoliumbromide (MTT) assay, and (c) the lactate dehydrogenase (LDH) assay; (2) glutathione redox state; and (3) glutathione peroxidase and glutathione reductase activities using a repeated measures experimental design. Results. At concentrations of 0.375 mg/mL and 0.5 mg/mL, the extract increased the intracellular levels of total and reduced glutathione and had no effect on the intracellular amounts of oxidized glutathione. The extract had no effect on glutathione peroxidase and glutathione reductase activities. Conclusion. These data indicate that the mechanism of the hepatoprotective action of aqueous extracts of T. polium may be, in part, due to augmenting intracellular glutathione levels.

Characterization of nonthermal focused ultrasound for noninvasive selective fat cell disruption (lysis): technical and preclinical assessment.

BACKGROUND: There has been a lack of published bench and preclinical data supporting the safety and effectiveness of a noninvasive, nonthermal, focused ultrasound technology for body contouring. METHODS: A series of experiments were conducted with a transcutaneous focused ultrasound device (UltraShape Contour I; UltraShape Inc., Yoqneam, Israel): (1) three-dimensional acoustic field distribution was measured by hydrophone in a water bath; (2) the real-time two-dimensional acoustic field was assessed using optical visualization by a Schlieren system; (3) three-dimensional and Schlieren results were compared in tissue-mimicking gel phantoms and in frozen specimens of porcine subcutaneous fat; and (4) a porcine in vivo preclinical model was used to test safety, selectivity, and efficacy by histological staining of excised skin and subcutaneous fat specimens. RESULTS: Real-time imaging of acoustic field distribution obtained by the Schlieren system, as well as real-time ultrasound visualization, produced stable cavitation both in water and in the gel phantoms. The area where the effect was visible corresponded to the focal area of energy delivered by the system transducer, as measured by the hydrophone. Histologically stained specimens of skin and subcutaneous fat that were excised from porcine studies (n = 14) following treatments (n = 31) demonstrated fat cell lysis and no observable cellular destruction of adjacent blood vessels, nerves, and connective tissue. No epidermal or dermal changes were observed clinically or histologically. CONCLUSIONS: The delivery of noninvasive focused ultrasonic energy has been validated and supported by the basic and preclinical data presented. Future studies will investigate various treatment regimens for improved body-contouring results.