Development of multicellular organisms requires the differential usage of our genetic information to change one cell fate into another. This process drives the appearance of different cell types that ...come together to form specialized tissues sustaining a healthy organism. In the last decade, by moving away from studying single genes toward a global view of gene expression control, a revolution has taken place in our understanding of how genes work together and how cells communicate to translate the information encoded in the genome into a body plan. The development of hematopoietic cells has long served as a paradigm of development in general. In this review, we highlight how transcription factors and chromatin components work together to shape the gene regulatory networks controlling gene expression in the hematopoietic system and to drive blood cell differentiation. In addition, we outline how this process goes astray in blood cancers. We also touch upon emerging concepts that place these processes firmly into their associated subnuclear structures adding another layer of the control of differential gene expression.
Hematopoiesis has long served as model to study the molecular mechanisms driving developmental processes in general. Here, we review how transcription factors and chromatin components cooperate within specific subnuclear structures to shape the gene regulatory networks controlling gene expression and differentiation in response to extracellular signals. We also review studies describing how gene regulatory networks are altered in blood cancers carrying regulator mutations and how such studies inform novel therapies.
Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we ...introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons”) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.
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•Plug-and-play reagents for gene- and cell-type-specific transgene expression in vivo•Binary and intersectional targeting is enabled for multiple transcriptional factors•Trojan exons couple to “off-the-shelf” Drosophila stocks bearing intronic MiMIC transposons•T-GEM Trojan exon can be used with the Crispr/Cas system to target introns in arbitrary genes of interest
Genetic manipulation and monitoring of cellular function in living animals requires tools for targeting transgene expression to specific cell types. Diao et al. present a “Trojan exon” approach that removes a common bottleneck in transgene targeting by facilitating the rapid production of transgenic organisms that can express transcriptional effectors in targeted cell types. The method uses off-the-shelf components in Drosophila and is modular, simple, fast, and precise.
Reproduction induces increased food intake across females of many animal species
, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the ...diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain.
In Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest by placing the Gal4 transcription factor under control of the gene's DNA regulatory ...elements. If these regulatory elements are not known, however, expression of Gal4 in the desired pattern may be difficult or impossible. To solve this problem, we have developed a method for co-expressing Gal4 with the endogenous gene by exploiting the "ribosomal skipping" mechanism of the viral T2A peptide. This method requires explicit knowledge only of the endogenous gene's open reading frame and not its regulatory elements.
The Trojan exon method, which makes use of intronically inserted T2A-Gal4 cassettes, has been widely used in
to create thousands of gene-specific Gal4 driver lines. These dual-purpose lines provide ...genetic access to specific cell types based on their expression of a native gene while simultaneously mutating one allele of the gene to enable loss-of-function analysis in homozygous animals. While this dual use is often an advantage, the truncation mutations produced by Trojan exons are sometimes deleterious in heterozygotes, perhaps by creating translation products with dominant negative effects. Such mutagenic effects can cause developmental lethality as has been observed with genes encoding essential transcription factors. Given the importance of transcription factors in specifying cell type, alternative techniques for generating specific Gal4 lines that target them are required. Here, we introduce a modified Trojan exon method that retains the targeting fidelity and plug-and-play modularity of the original method but mitigates its mutagenic effects by exploiting the self-splicing capabilities of split inteins. "Split Intein Trojan exons" (siTrojans) ensure that the two truncation products generated from the interrupted allele of the native gene are trans-spliced to create a full-length native protein. We demonstrate the efficacy of siTrojans by generating a comprehensive toolkit of Gal4 and Split Gal4 lines for the segmentally expressed Hox transcription factors and illustrate their use in neural circuit mapping by targeting neurons according to their position along the anterior-posterior axis. Both the method and the Hox gene-specific toolkit introduced here should be broadly useful.
The shedding of the old exoskeleton that occurs in insects at the end of a molt (a process called ecdysis) is typically followed by the expansion and tanning of a new one. At the adult molt, these ...postecdysial processes include expansion and hardening of the wings. Here we describe recent advances in understanding the neural and hormonal control of wing expansion and hardening, focusing on work using Drosophila melanogaster in which genetic manipulations have permitted detailed investigation of postecdysial processes and their modulation by sensory input. To place this work in context, we briefly review recent progress in understanding the neuroendocrine regulation of ecdysis, which appears to be largely conserved across insect species. Investigations into the neuroendocrine networks that regulate ecdysial and postecdysial behaviors provide insights into how stereotyped, yet environmentally responsive, sequences are generated and how they develop and evolve.
Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream.
Here we report a novel ...function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts—beyond its known roles in development—as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2, the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP.
We therefore identify a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells.
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•Enteroendocrine cells control the local stem cell niche via Bursicon•Enteroendocrine cells express the Bursicon ligand, and the muscle its receptor, DLGR2•Bursicon/DLGR2 signaling increases cAMP in visceral muscle to control EGF-like Vein•Bursicon signaling in the adult midgut instructs intestinal stem cell quiescence
Scopelliti et al. show that in the adult Drosophila midgut, enteroendocrine cells act locally—using Bursicon/DLGR2 signaling—to restrain stem cell proliferation via paracrine signaling to the mesenchymal stem cell niche.
Many visual animals have innate preferences for particular wavelengths of light, which can be modified by learning. Drosophila's preference for UV over visible light requires UV-sensing R7 ...photoreceptors and specific wide-field amacrine neurons called Dm8. Here we identify three types of medulla projection neurons downstream of R7 and Dm8 and show that selectively inactivating one of them (Tm5c) abolishes UV preference. Using a modified GRASP method to probe synaptic connections at the single-cell level, we reveal that each Dm8 neuron forms multiple synaptic contacts with Tm5c in the center of Dm8's dendritic field but sparse connections in the periphery. By single-cell transcript profiling and RNAi-mediated knockdown, we determine that Tm5c uses the kainate receptor Clumsy to receive excitatory glutamate input from Dm8. We conclude that R7s→Dm8→Tm5c form a hard-wired glutamatergic circuit that mediates UV preference by pooling ∼16 R7 signals for transfer to the lobula, a higher visual center.
Additive manufacturing of copper enables enhanced design freedom which allows for improved performance of components in thermal management and electrical applications. Joining via soldering provides ...ideal electrical and thermal connections, but the solderability of complex additively manufactured surfaces is poorly understood. In the present work, the solderability of nominally pure copper coupons manufactured by three additive manufacturing techniques (laser powder bed fusion, laser engineered net shaping, and bound powder extrusion) was experimentally assessed using the wetting balance technique and pin pull testing. Coupons produced by each method were tested as built and after surface modification by dry electropolishing. Contact angles and wetting times were calculated from wettability testing. Peak tensile loads required to remove pins soldered to coupons were also recorded for each surface condition. The dipped coupons and solder joint fracture surfaces were examined with optical and scanning electron microscopes. It was found that nonuniform wetting and excessive wicking of solder can result in weak joints, and surface modification positively affected overall solderability in all cases. All surfaces were shown to be wettable, but bound powder extrusion was found to produce the most solderable copper surfaces among the additive manufacturing methods tested.
Radio waves are highly penetrating, non-ionizing, and cause minimal damage to surrounding tissues. Radio wave control of drug release has been achieved using a novel thermoresponsive copolymer bound ...to a superparamagnetic iron oxide nanoparticle (SPION) core. A NIPAM-acrylamide-methacrolein copolymer underwent a coil-to-globular structure phase change upon reaching a critical temperature above the human body temperature but below hyperthermic temperatures. The copolymer was covalently bound to SPIONs which increase in temperature upon exposure to radio waves. This effect could be controlled by varying input energies and frequencies. For controlled drug release, proteins were bound via aldehyde groups on the copolymer and amine groups on the protein. The radio wave-induced heating of the complex thereby released the drug-bearing proteins. The fine-tuning of the radio wave exposure allowed multiple cycles of protein-drug release. The fluorescent tagging of the complex by FITC was also achieved in situ, allowing the tagging of the complex. The localization of the complex could also be achieved in vitro under a permanent magnetic field.
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