Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.

/pdf/a-genome-wide-transgenic-rnai-library-for-conditional-gene-20svjywsa1.pdf

A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

MicroRNAs have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that some microRNAs can function as oncogenes or tumour suppressors, the role of microRNAs in mediating cancer metastasis remains unexplored. Here we show, using a combination of mouse and human cells, that microRNA-10b (miR-10b) is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. Overexpression of miR-10b in otherwise non-metastatic breast tumours initiates robust invasion and metastasis. Expression of miR-10b is induced by the transcription factor Twist, which binds directly to the putative promoter of mir-10b (MIRN10B). The miR-10b induced by Twist proceeds to inhibit translation of the messenger RNA encoding homeobox D10, resulting in increased expression of a well-characterized pro-metastatic gene, RHOC. Significantly, the level of miR-10b expression in primary breast carcinomas correlates with clinical progression. These findings suggest the workings of an undescribed regulatory pathway, in which a pleiotropic transcription factor induces expression of a specific microRNA, which suppresses its direct target and in turn activates another pro-metastatic gene, leading to tumour cell invasion and metastasis.

Tumour invasion and metastasis initiated by microRNA-10b in breast cancer

https://www.valadkhanlab.org/599_lectures/Khalil/A%20Long%20Noncoding%20RNA%20Controls%20Muscle%20Differentiation%20by%20Functioning%20as%20a%20Competing%20Endogenous%20RNA.pdf

A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA

The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (gamma-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus.

/pdf/leptin-activates-anorexigenic-pomc-neurons-through-a-neural-u14hlek2a4.pdf

Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus

We have developed a method for temporal and regional gene expression targeting (TARGET) in Drosophila and show the simultaneous spatial and temporal rescue of a memory defect. The transient expression of the rutabaga-encoded adenylyl cyclase in the mushroom bodies of the adult brain was necessary and sufficient to rescue the rutabaga memory deficit, which rules out a developmental brain defect in the etiology of this deficit and demonstrates an acute role for rutabaga in memory formation in these neurons. The TARGET system offers general utility in simultaneously addressing issues of when and where gene products are required.

https://science.sciencemag.org/content/sci/302/5651/1765.full.pdf

Spatiotemporal rescue of memory dysfunction in Drosophila

Memories are thought to be due to lasting synaptic modifications in the brain. The search for memory traces has relied predominantly on determining regions that are necessary for the process. However, a more informative approach is to define the smallest sufficient set of brain structures. The rutabaga adenylyl cyclase, an enzyme that is ubiquitously expressed in the Drosophila brain and that mediates synaptic plasticity, is needed exclusively in the Kenyon cells of the mushroom bodies for a component of olfactory short-term memory. This demonstrates that synaptic plasticity in a small brain region can be sufficient for memory formation.

https://science.sciencemag.org/content/sci/288/5466/672.full.pdf

Localization of a short-term memory in Drosophila.

Members of the myocyte enhancer binding factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, and serum response factor) box transcription factors are expressed in the skeletal, cardiac, and smooth muscle lineages of vertebrate and Drosophila embryos. These factors bind an adenine-thymidine-rich DNA sequence associated with muscle-specific genes. The function of MEF2 was determined by generating a loss-of-function of the single mef2 gene in Drosophila (D-mef2). In loss-of-function embryos, somatic, cardiac, and visceral muscle cells did not differentiate, but myoblasts were normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2.

Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila

Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.

Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development

The myocyte enhancer factor (MEF) 2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates. We have cloned a protein from Drosophila, termed D-MEF2, that shares extensive amino acid homology with the MADS (MCM1, Agamous, Deficiens, and serum-response factor) domains of the vertebrate MEF2 proteins. D-mef2 gene expression is first detected during Drosophila embryogenesis within mesodermal precursor cells prior to specification of the somatic and visceral muscle lineages. Expression of D-mef2 is dependent on the mesodermal determinants twist and snail but independent of the homeobox-containing gene tinman, which is required for visceral muscle and heart development. D-mef2 expression precedes that of the MyoD homologue, nautilus, and, in contrast to nautilus, D-mef2 appears to be expressed in all somatic and visceral muscle cell precursors. Its temporal and spatial expression patterns suggest that D-mef2 may play an important role in commitment of mesoderm to myogenic lineages.

https://www.pnas.org/content/pnas/91/12/5662.full.pdf

Robert A. Schulz

Papers

Localization of a short-term memory in Drosophila.

Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila

Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors.

Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development

D-MEF2: A MADS box transcription factor expressed in differentiating mesoderm and muscle cell lineages during Drosophila embryogenesis