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In Drosophila, dopaminergic (DA) neurons can be found from mid embryonic stages of development till adulthood. Despite their functional involvement in learning and memory, not much is known about the developmental as well as molecular mechanisms involved in the events of DA neuronal specification, differentiation and maturation. In this report we demonstrate that most larval DA neurons are generated during embryonic development. Furthermore, we show that loss of function (l-o-f) mutations of genes of the apical complex proteins in the asymmetric cell division (ACD) machinery, such as inscuteable and bazooka result in supernumerary DA neurons, whereas l-o-f mutations of genes of the basal complex proteins such as numb result in loss or reduction of DA neurons. In addition, when Notch signaling is reduced or abolished, additional DA neurons are formed and conversely, when Notch signaling is activated, less DA neurons are generated. Our data demonstrate that both ACD and Notch signaling are crucial mechanisms for DA neuronal specification. We propose a model in which ACD results in differential Notch activation in direct siblings and in this context Notch acts as a repressor for DA neuronal specification in the sibling that receives active Notch signaling. Our study provides the first link of ACD and Notch signaling in the specification of a neurotransmitter phenotype in Drosophila. Given the high degree of conservation between Drosophila and vertebrate systems, this study could be of significance to mechanisms of DA neuronal differentiation not limited to flies.

Mesenchymal stem cells (MSCs) have multilineage differentiation potential which includes cell lineages of the central nervous system; hence MSCs might be useful in the treatment of neurodegenerative diseases such as Parkinson's disease. Although mesenchymal stem cells have been shown to differentiate into the neural lineage, there is still little knowledge about the underlying mechanisms of differentiation particularly towards specialized neurons such as dopaminergic neurons. Here, we show that MSCs derived from human umbilical cord blood (MSC(hUCBs)) are capable of expressing tyrosine hydroxylase (TH) and Nurr1, markers typically associated with DA neurons. We also found differential phosphorylation of TH isoforms indicating the presence of post-translational mechanisms possibly activating and modifying TH in MSC(hUCB). Furthermore, functional dissection of components in the differentiation medium revealed that dibutyryl-cAMP (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX) and retinoic acid (RA) are involved in the regulation of Nurr1 and Neurofilament-L expression as well as in the differential phosphorylation of TH. We also demonstrate a possible inhibitory role of the protein kinase A signaling pathway in the phosphorylation of specific TH isoforms.

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.

Mesenchymal stem cells (MSC) are multipotent and give rise to distinctly differentiated cells from all three germ layers. While umbilical cord blood derived mesenchymal-like cells were previously shown to be capable of differentiating into the neural lineage both in vitro and in vivo, the underlying molecular mechanisms and signal transduction pathways remain to be elucidated. In this study, we show that mesenchymal-like cells from umbilical cord blood are capable of neural differentiation and this capability is mediated by the Protein kinase A (PKA) signal transduction pathway. While activation of PKA via experimental cAMP upregulation leads to outgrowth of neurite-like structures as well as expression of neural marker genes, blocking PKA activity completely abolishes all these features. Thus, our results demonstrate that PKA function is sufficient and required for neurite-like outgrowth and regulation of neural specific gene expression in mesenchymal-like stem cells from cord blood.

Asymmetric cell divisions can be mediated by the preferential segregation of intrinsic cell fate determinants into one of two sibling daughters. In dividing Drosophila neural progenitors the apical-basal orientation of the mitotic spindle, the basal cortical localization of the cell fate determinants Numb and/or Prospero as well as the coordination of these events are mediated by several proteins which include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins) which localize as an apical cortical complex starting at interphase. Here I will summarize data which suggest that the formation of this apical complex involves two distinct steps: (1) during the initiation of apical complex formation in interphase neuroblasts, there appears to be a hierarchical relation amongst these components where Baz recruits Insc and Baz/Insc in turn recruit Pins to the apical cortex/stalk; (2) while in delaminated mitotic neuroblasts the maintenance of the apical cortical localization of these proteins is dependent on the presence of all three components. Moreover, we show that the maintenance of this apical protein complex is essential for the correct execution of asymmetric division. Finally, the localization of the various asymmetrically localized proteins shows cell cycle dependence; however, the involvement of the cell cycle regulator in asymmetric cell divisions has not been previously shown. Here we present evidence from ongoing experiments which suggest a requirement for the key cell cycle regulator cdc2 in asymmetric cell divisions.