Notch signaling and the generation of cell diversity in Drosophila neuroblast lineages.

Notch is a membrane bound transcription factor and it plays fundamental roles in many cell-cell interaction events usually involving directly neighboring cells relating an extrinsic signal of a sending cell to the nucleus of the receiving cell to modulate gene expression patterns in this cell. Notch regulates cell fate specification, cell proliferation as well as cell death in the contexts of many organs and cell types. Although the mechanisms of signal transduction from the cell surface to the nucleus are relatively simple, it is not fully understood how such a straightforward pathway can result in tremendously complex outcomes at the cellular level. This chapter discusses some of the known roles of Notch during central nervous system development in Drosophila. In the CNS, Notch is a major player in creating cellular diversity on the level of binary cell fates by possibly activating differential gene expression in sibling cells arising through asymmetric cell division. This chapter also raises some questions related to Notch function during neural cell fate specification which have not yet been satisfactorily addressed in the field. Finding answers to these questions may provide further insights into how cell-cell interactions in the nervous system involving Notch control the generation of cellular diversity.

The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila.

During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC). We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.

Asymmetric cell division and Notch signaling specify dopaminergic neurons in Drosophila.

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.

Orthodenticle is necessary for survival of a cluster of clonally related dopaminergic neurons in the Drosophila larval and adult brain.

BACKGROUND: The dopaminergic (DA) neurons present in the central brain of the Drosophila larva are spatially arranged in stereotyped groups that define clusters of bilaterally symmetrical neurons. These clusters have been classified according to anatomical criteria (position of the cell bodies within the cortex and/or projection pattern of the axonal tracts). However, information pertaining to the developmental biology, such as lineage relationship of clustered DA neurons and differential cell subtype-specific molecular markers and mechanisms of differentiation and/or survival, is currently not available. RESULTS: Using MARCM and twin-spot MARCM techniques together with anti-tyrosine hydroxylase immunoreactivity, we have analyzed the larval central brain DA neurons from a developmental point of view and determined their time of birth, their maturation into a DA neurotransmitter phenotype as well as their lineage relationships. In addition, we have found that the homeodomain containing transcription factor Orthodenticle (Otd) is present in a cluster of clonally related DA neurons in both the larval and adult brain. Taking advantage of the otd hypomorphic mutation ocelliless (oc) and the oc2-Gal4 reporter line, we have studied the involvement of orthodenticle (otd) in the survival and/or cell fate specification of these post-mitotic neurons. CONCLUSIONS: Our findings provide evidence of the presence of seven neuroblast lineages responsible for the generation of the larval central brain DA neurons during embryogenesis. otd is expressed in a defined group of clonally related DA neurons from first instar larvae to adulthood, making it possible to establish an identity relationship between the larval DL2a and the adult PPL2 DA clusters. This poses otd as a lineage-specific and differential marker of a subset of clonally related DA neurons. Finally, we show that otd is required in those DA neurons for their survival.

Fetomaternal microchimerism: Some answers and many new questions.

The transfer of fetal cells into mothers during pregnancy and their organ specific integration is a well recognized phenomenon in placental vertebrates. Recently, it has been reported that some fetal cells found in the mothers have progenitor cell-like features such as multilineage differentiation potential and as a consequence they were termed pregnancy associated progenitor cells (PAPC). The multilineage differentiation potential suggested some level of cellular plasticity, which these cells share with other stem or progenitor cells. In this context, we have shown that PAPCs indeed express neural stem cell and markers for developing neurons in the brain and that PAPCs morphologically mature into neurons over time. The stem/progenitor properties of PAPCs raises the hope that they might be valuable for studying the functional integration of foreign cells into preexisting tissues and organs, for example in cellular therapies. The functional integration of transplanted cells and their connectivity to the host circuitry is still a major bottleneck in cellular therapies particularly for the brain. The animal models of fetomaternal microchimerism might provide valuable insights into the mechanism how cells survive, migrate, integrate and differentiate in a foreign environment of a host. This review discusses some of the recent findings in the field of fetomaternal microchimerism. It also tries to identify some major gaps of knowledge and raises some questions resulting from the recent advances. Studying fetomaternal microchimerism and the properties of PAPCs in greater detail might pave the way to advance cell based regenerative medicine as well as transplantation medicine.

Pregnancy-associated progenitor cells differentiate and mature into neurons in the maternal brain.

Bidirectional cell trafficking between fetus and mother during pregnancy is a well-established phenomenon observed in placental vertebrates including humans. Although studies have shown that transmigratory fetal cells, also termed pregnancy-associated progenitor cells (PAPCs), can integrate into multiple maternal organs, the integration, long-term survival, and differentiation of PAPCs in the brain has not been extensively studied. Using a murine model of fetomaternal microchimerism, we show that PAPCs integrated and persisted in several areas of the maternal brain for up to 7 months postpartum. Besides expressing neural stem cell or immature neuronal markers, PAPCs were observed to express mature neuronal markers, indicating that PAPCs adopted a neuronal fate. Further, PAPCs also displayed morphologically neuronal maturation by an increasing axonal/dendritic complexity over time. Therefore, PAPCs seem to undergo a molecular and morphological maturation program similar to that observed during adult neurogenesis. We provide evidence that neuronal gene expression of PAPCs was not a consequence of cell fusion with maternal neurons. In addition, in mothers with experimentally induced Parkinson's disease (PD), the frequency of PAPCs within the hippocampus initially increased whereas long-term presence of PAPCs was compromised. Also, the spatial distribution of PAPCs within the hippocampus was altered in mothers with PD. Thus, the disease context influenced the initial attraction, long-term survival, and spatial distribution of PAPCs, which may have wider implications on cell replacement strategies in human neurodegenerative diseases such as PD.

Genetic interactions of eyes absent, twin of eyeless and orthodenticle regulate sine oculis expression during ocellar development in Drosophila.

The homeobox gene sine oculis (so) is required for the development of the entire visual system in Drosophila, which includes the compound eyes, the ocelli, the optic lobe of the brain and the Bolwig's organ. During ocelli development, so expression labels, together with eyes absent (eya), the emergence of the ocellar precursor cells in the third instar eye-antennal disc. Footprinting and misexpression studies have led to the proposal that the Pax6 homologue twin of eyeless (toy) directly regulates the initiation of so expression in ocellar precursor cells. However, so expression in a toy loss-of-function mutant background has not been yet analyzed due to the lack of eye-antennal disc development in strong toy mutant alleles. Using an embryonic eye primordium-specific enhancer of toy, we have rescued the developmental defect of a strong toy mutant allele and analyzed so expression in the ocelli primordium of toy loss-of-function eye-antennal discs during third instar larva. The results show that so expression is only marginally affected in the absence of Toy transcriptional activity and that the toy positive effect on so expression is largely eya-mediated. These results suggest that eya is the main factor controlling both initiation and maintenance of so expression in ocellar precursor cells. In addition, we present the characterization of a new minimal eye/ocellus-specific enhancer of the so gene.

Roles of db-cAMP, IBMX and RA in aspects of neural differentiation of cord blood derived mesenchymal-like stem cells.

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.

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages.

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.

MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.

Inscuteable-independent apicobasally oriented asymmetric divisions in the Drosophila embryonic CNS.

Inscuteable is the founding member of a protein complex localised to the apical cortex of Drosophila neural progenitors that controls their asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterised to date, in both the central and peripheral nervous systems, require inscuteable. Here we examine the generality of this requirement. We show that many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, we focused on the MP2 > dMP2 + vMP2 division. We show that for MP2 divisions, asymmetric localisation and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. We conclude that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable.