Molecular profiling of stem cell-like female germ line cells in Drosophila delineates networks important for stemness and differentiation.

Stem cells can self-renew and produce daughter cells destined for differentiation. The precise control of the balance between these two outcomes is essential to ensure tissue homeostasis and to prevent uncontrolled proliferation resulting in tumor formation. As self-renewal and differentiation are likely to be controlled by different gene expression programs, unraveling the underlying gene regulatory networks is crucial for understanding the molecular logic of this system. In this study, we have characterized by next generation RNA sequencing (RNA-seq) the transcriptome of germline stem cell (GSC)-like cells isolated from bag of marbles (bam) mutant Drosophila ovaries and compared it to the transcriptome of germ line cells isolated from wild-type ovaries. We have complemented this dataset by utilizing an RNA-immunoprecipitation strategy to identify transcripts bound to the master differentiation factor Bam. Protein complex enrichment analysis on these combined datasets allows us to delineate known and novel networks essential for GSC maintenance and differentiation. Further comparative transcriptomics illustrates similarities between GSCs and primordial germ cells and provides a molecular footprint of the stem cell state. Our study represents a useful resource for functional studies on stem cell maintenance and differentiation.

Myc and the Tip60 chromatin remodeling complex control neuroblast maintenance and polarity in Drosophila.

Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. We addressed this question in Drosophila neural stem cells called neuroblasts. We identified the Tip60 chromatin remodeling complex and its interaction partner Myc as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. We found that aPKC is the key target gene of Myc and the Tip60 complex subunit Domino in regulating neuroblast polarity. Our transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. Our findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division.

Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells.

Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz(4) and baz(815-8) alleles with those of the so far uncharacterized baz(XR11) and baz(EH747) null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in baz(EH747) and baz(XR11) while baz(4) and baz(815) (-8) show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz(4) and baz(815-8) alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type.

Flow cytometry-based evaluation and enrichment of multiwalled carbon nanotube dispersions.

The uniform aqueous dispersion of carbon nanotubes (CNTs) is a vital but challenging task required for their utilization in most technologies. We propose and demonstrate a technique based on forward- and side-scatter analysis on a flow cytometer to characterize the components in a dispersion of multiwalled CNTs (MWCNTs). The method simultaneously distinguishes various MWCNT components such as short and long CNTs, nanotube bundles, and particulates. It also detects the emergence of new CNT populations as a result of centrifugation. We use this method, together with classical methods such as UV and Raman spectroscopy, to observe and study the multistep MWCNT dispersion process in various surfactants (Pluronic, Triton X-100, sodium dodecyl sulfate, and cetyl trimethylammonium bromide). On the basis of the distinct scatter patterns obtained, we confirm and elaborate the surfactant-assisted unzipping mechanism of MWCNT dispersion. We also show that the ultrasonic energy spent after MWCNT unbundling and unwinding can be minimized and the process optimized for each surfactant by correct end point detection through scatter analysis. The ability to enrich nanotube population in dispersion by using the sorting mode of a flow cytometer is confirmed by electron microscopy and Raman spectroscopy. This method can thus be used for observing and enriching MWCNT components and as a complementary technique to UV spectroscopy for studying and optimizing MWCNT dispersion in surfactants.

All-trans retinoic acid loaded block copolymer nanoparticles efficiently induce cellular differentiation in HL-60 cells.

All-trans retinoic acid (atRA) is used in the differentiation therapy of Acute Promyelocytic Leukemia. However, its therapeutic success is limited by the appearance of relapse and recalcitrant cases, poor aqueous solubility and high degradability. In the current work, we prepared two types of atRA-loaded copolymer nanoparticles - PL1RA and PC1RA, based on poly(ethyleneglycol) (PEG)-poly(l-lactide) and PEG-poly(ε-caprolactone), respectively. We then evaluated their physico-chemical properties and compared their differentiation-inducing potential of HL-60 cells with free atRA. These nanoparticles were in the size range 100-150nm, possessed moderate colloidal stability and exhibited around 30% encapsulation efficiencies. In vitro release studies indicated pseudo-zero order release of a sustained nature, with PL1RA showing 71% and PC1RA exhibiting 84% drug release over a period of two weeks. Photostability measurements exhibited considerable increase in atRA photostability in the nanoparticle forms: 25% of the drug in PL1RA and 19% in PC1RA was intact as compared to only 5% for free atRA after 8h of light exposure. PL1RA and PC1RA exhibited efficacies comparable to free atRA in inducing HL-60 respiratory burst as assessed by nitroblue tetrazolium and 2',7'-dichlorodihydro fluorescein diacteate assays. The average CD11b expressions for atRA, PL1RA and PC1RA on day 5 of treatment were 58%, 49% and 60%, respectively. Post-differentiation apoptosis (∼40%) and reduction in mitochondrial transmembrane potentials (∼60-70%) were also comparable across all treatment groups. Therefore, our block copolymer nanoparticles, PL1RA and PC1RA, are attractive and effective vehicles for atRA delivery which maintain its activity and enhance its stability resulting in efficient induction of HL-60 differentiation.

Synthesis of BF2 complexes of prodigiosin type oligopyrroles.

We developed a simple, facile route for the synthesis of BF(2) complexes of prodigiosin type oligopyrroles and their cholesterol conjugates. This route gives an access to synthesize any desired meso-aryl-substituted 3-pyrrolyl BODIPYs which were not easily accessible earlier.

Flow cytometry as a novel tool to evaluate and separate vesicles using characteristic scatter signatures.

Vesicles are usually characterized for their structure by microscopy or, less often, by the addition of fluorescent dyes in a flow cytometer. We present a new method of studying these structures and associated forms by forward and side scatter analysis on a flow cytometer which has the advantage of simultaneous handling of large population of vesicles to identify their shapes and lamellarities. The technique is suitable for several types of vesicular structures like Multivesicular vesicles (MVV), multilamellar and unilamellar vesicles. Characteristic signatures are given by tubular structures and fine features thereon allow detection of complex structures such as fused and ellipsoidal forms. Coexistence of tubular and spherical structures, such as those known to form when surfactants/salt solutions are diluted, can clearly be detected by the signature pattern, which separates into two distinctly identifiable populations. The population can be sorted or separated easily based on these signatures and such sorting has allowed us to confirm our findings by microscopic observations. This novel method can thus be used for concurrent observations of vesicle populations with dye or more advantageously without employing any fluorescent tag.