Neuronal diversity and convergence in a visual system developmental atlas.

Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains1,2, the large neuronal diversity of the Drosophila optic lobe3 and its connectome4-6 are almost completely characterized. However, a molecular characterization of this neuronal diversity, particularly during development, has been lacking. Here we present insights into brain development through a nearly complete description of the transcriptomic diversity of the optic lobes of Drosophila. We acquired the transcriptome of 275,000 single cells at adult and at five pupal stages, and built a machine-learning framework to assign them to almost 200 cell types at all time points during development. We discovered two large neuronal populations that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signalling throughout development. Moreover, we show that the transcriptomes of neurons that are of the same type but are produced days apart become synchronized shortly after their production. During synaptogenesis we also resolved neuronal subtypes that, although differing greatly in morphology and connectivity, converge to indistinguishable transcriptomic profiles in adults. Our datasets almost completely account for the known neuronal diversity of the Drosophila optic lobes, and serve as a paradigm to understand brain development across species.

Phenotypic Convergence: Distinct Transcription Factors Regulate Common Terminal Features.

Transcription factors regulate the molecular, morphological, and physiological characteristics of neurons and generate their impressive cell-type diversity. To gain insight into the general principles that govern how transcription factors regulate cell-type diversity, we used large-scale single-cell RNA sequencing to characterize the extensive cellular diversity in the Drosophila optic lobes. We sequenced 55,000 single cells and assigned them to 52 clusters. We validated and annotated many clusters using RNA sequencing of FACS-sorted single-cell types and cluster-specific genes. To identify transcription factors responsible for inducing specific terminal differentiation features, we generated a "random forest" model, and we showed that the transcription factors Apterous and Traffic-jam are required in many but not all cholinergic and glutamatergic neurons, respectively. In fact, the same terminal characters often can be regulated by different transcription factors in different cell types, arguing for extensive phenotypic convergence. Our data provide a deep understanding of the developmental and functional specification of a complex brain structure.

Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion.

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second-order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)-derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3-L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion. Copyright © 2014 John Wiley & Sons, Ltd.

Neural stem cells from mouse strain Thy1 YFP-16 are a valuable tool to monitor and evaluate neuronal differentiation and morphology.

To analyse events following transplantation of stem cells in the brain robust tools for tracing stem cells are required. Here we took advantage of the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy1 YFP-16), where yellow fluorescent protein (YFP) is under control of the promoter of Thy1 gene. This allows visualising whole neurons, i.e. their cell body, axons and dendrites. In this work fluorescent cells were followed during embryonic development, in vitro differentiation, and after transplantation in the healthy and stroke-affected mouse brain. During embryonic development Thy1-YFP positive cells were first observed on E12.5 and subsequently located in the prosencephalon, rhombencephalon, spinal cord and peripheral nerves. Quantitative analysis by RT-PCR and immunocytochemistry revealed that Thy1-YFP positive cells during embryo development and in vitro differentiation were expressing nestin and SOX2 then MAP2, ß3-tubulin and NeuN. Thy1-YFP positive cells isolated from E14.5 represented 21.88±053% (SD) of the cultivated neurons and this remained constant along in vitro differentiation. On the other hand, proportion of Thy1-YFP positive cells reached 50% of neurons in perinatal and one month old mouse brain. Neural stem cells isolated from Thy1 YFP-16 mouse strain transplanted near hippocampus of the healthy and stroke-affected brain were distinguishable by YFP fluorescence. They differentiated into mature neurons and were detectable even 14 weeks after transplantation, the end point of our experiment. In conclusion, stem cells originating from Thy1 YFP-16 mice represent an outstanding tool to monitor neurogenesis enabling morphological analyses of new neurons and their projections, in particular after transplantation in the brain.

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.

Krüppel-like transcription factor 8 (Klf8) is expressed and active in the neurons of the mouse brain.

Krüppel-like transcription factor 8 (KLF8) is a transcription factor suggested to be involved in various cellular events, including malignant cell transformation, still its expression in the adult rodent brain remained unknown. To analyze Klf8 in the mouse brain and to identify cell types expressing it, a specific transgenic Klf8(Gt1Gaj) mouse was used. The resulting Klf8 gene-driven ß-galactosidase activity was visualized by X-gal histochemical staining of the brain sections. The obtained results were complemented by in situ RNA hybridization and immunohistochemistry. Klf8 was highly expressed throughout the adult mouse brain gray matter including the cerebral cortex, hippocampus, olfactory bulb, hypothalamus, pallidum, and striatum, but not in the cerebellum. Immunofluorescent double-labeling revealed that KLF8-immunoreactive cells were neurons, and the staining was located in their nucleus. This was the first study showing that Klf8 was highly expressed in various regions of the mouse brain and in particular in the neurons, where it was localized in the cell nuclei.

Immunohistochemical expression of STAM2 in gastrointestinal stromal tumors.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract, believed to originate from the interstitial cells of Cajal or their stem cell-like precursors. Recent studies incidentally found the expression in interstitial cells of Cajal of the signal-transducing adaptor molecule-2 (STAM2), which is an endosomal protein acting as a regulator of receptor signaling and trafficking. Here, we investigated the immunohistochemical expression of STAM2 in GIST. MATERIALS AND METHODS: To evaluate the level of STAM2 expression, the percentage of cells staining positively for STAM2 and their staining intensity were graded on a scale of 0-3 and then multiplied to give the staining index as: 0=none; 1-3=low; 4-6=moderate and 9=high. RESULTS: In 51 analyzed GIST samples, expression of STAM2 was observed in 45 cases (88.2%). Based on antibody screening, we observed a positive correlation between the expression of GIST marker stem cell growth factor receptor, also known as tyrosine-protein kinase KIT or CD117, and STAM2 expression (r=0.387, p<0.003). To identify possible STAM2 function in GIST, we performed correlation analysis between STAM2 expression and tumor size, primary tumor site, tumor type, mitotic count, Ki-67 proliferative index, risk stratification and development of recurrent/metastatic disease. Among these parameters, only correlation between the percentage of STAM2-positive cells and mitotic count was statistically significant (r=-0.362, p<0.01). CONCLUSION: Further studies are required to unravel the role of STAM2 in the oncogenic cell phenotype of GIST.

Neurons and a subset of interstitial cells of Cajal in the enteric nervous system highly express Stam2 gene.

Signal transducing adaptor molecule 2 (STAM2) is a phosphotyrosine protein, which is a member of the endosomal sorting complex required for transport (ESCRT-0) and is involved in the sorting process of the mono-ubiquitinated endosomal cargo for degradation in the lysosome. Analysis of gene trap mice carrying lacZ in frame with Stam2 revealed beta-galactosidase activity in the enteric nervous system (both in the myenteric and submucosal plexus) throughout the digestive tract. STAM2 immunostaining confirmed that the observed beta-galactosidase activity coincided with high Stam2 expression. To identify cell types with high Stam2 expression, STAM2 immunostaining was colocalized with the neuronal markers microtubule-associated protein 2 and protein gene product 9.5 and with c-kit as a marker for interstitial cells of Cajal (ICCs). STAM2 and c-kit positive cells comprised a subset of ICCs in the enteric nervous system. Qualitative and quantitative analysis of the morphology of the enteric nervous system in the homozygous mice carrying gene trap insertion in the Stam2 gene did not reveal phenotype changes; therefore, STAM2 function in the digestive tube remains elusive.

Stam2 expression pattern during embryo development.

STAM2 is a tyrosine-phosphorylated protein suggested to be involved in cargo selection during endocytic pathway, regulation of exocytosis and intracellular signaling. Gene trap method was used to create via insertional mutagenesis a mutant mouse line with integration of promoterless ßgeo (lacZ-neomycin phosphotransferase fusion) gene in the second intron of Stam2 gene, enabling analysis of its in vivo expression and function. The inserted ß-galactosidase (lacZ) reporter gene was used to reveal Stam2 expression during development. Stam2 in situ RNA hybridization and immunostaining confirmed the observed ß-galactosidase activity reflecting high Stam2 expression. The homozygous mutant mice showed no overt phenotypic alterations. Stam2 expression was detected after E9.5 in the gut, notochord, neural tube and heart. In the nervous system it was located in the floor, roof and basal plates of the developing neural tube, and in the developing cortex, hippocampus and olfactory bulbs. Toward the end of gestation, Stam2 expression appeared in the testis and ovary, lungs, nasal cavity epithelium, kidneys, urogenital sinus, intestine, pancreas, pituitary and adrenal glands, muscles, brown adipose tissue, skin and epithelium of the tongue and oral cavity.

Spectrophotometric analysis of flavonoid-DNA interactions and DNA damaging/protecting and cytotoxic potential of flavonoids in human peripheral blood lymphocytes.

The ability of luteolin, kaempferol and apigenin to bind to calf thymus (ct)-DNA, mode of action and stability of flavonoids in buffer were investigated. Spectrophotometric analysis revealed a rapid degradation of apigenin in an aqueous medium, while kaempferol and luteolin were stable for 24h upon dissolution in water. Spectrophotometric study of the interactions of kaempferol and luteolin with calf thymus DNA suggests classic intercalation as their dominant binding mode to DNA. Cytotoxicity/genotoxicity and cytoprotective/genoprotective effects of flavonoids in non-stressed and hydrogen peroxide stressed human peripheral lymphocytes were investigated using the fluorescent dye exclusion method and alkaline comet assay. Flavonoids revealed significant genoprotective effects in hydrogen peroxide stressed cells and in cells submitted to longer incubation in the cell culture medium. Luteolin, followed by apigenin and kaempferol, was shown to be the most effective in protecting DNA from oxidative damage induced by hydrogen peroxide. However, the investigated flavonoids also induced DNA damage, indicating their prooxidative capacity. The balance between the protection of DNA from oxidative damage and prooxidative effects was strongly dependent on flavonoid concentration and the incubation period.