The Glial Regenerative Response to Central Nervous System Injury Is Enabled by Pros-Notch and Pros-NFκB Feedback

Organisms are structurally robust, as cells accommodate changes preserving structural integrity and function. The molecular mechanisms underlying structural robustness and plasticity are poorly understood, but can be investigated by probing how cells respond to injury. Injury to the CNS induces proliferation of enwrapping glia, leading to axonal re-enwrapment and partial functional recovery. This glial regenerative response is found across species, and may reflect a common underlying genetic mechanism. Here, we show that injury to the Drosophila larval CNS induces glial proliferation, and we uncover a gene network controlling this response. It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NFκB. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment. Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness. This gene network may be of relevance to mammalian glia to promote repair upon CNS injury or disease

FACS purification and transcriptome analysis of drosophila neural stem cells reveals a role for Klumpfuss in self-renewal

Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well

Classification and nomenclature of all human homeobox genes

<p>Abstract</p> <p>Background</p> <p>The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.</p> <p>Results</p> <p>We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive <it>DUX1 </it>to <it>DUX5 </it>homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.</p> <p>Conclusion</p> <p>We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.</p

Drosophila dyskerin is required for somatic stem cell homeostasis

Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, we used this organism to investigate the specific role played by dyskerin in somatic stem cell maintenance. To this aim, we focussed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, a few dispersed precursors found in the depleted midguts can maintain stem identity and the ability to divide asymmetrically, nor show cell-growth defects or undergo apoptosis. Instead, their loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction

The identification of the Prox gene family and the development of optical projection tomography for use in Zebrafish

Vertebrate homologues of the Drosophila melanogaster gene prospero have been identified in many species. Whilst the function and regulation of prospero has been well studied in Drosophila the function and regulation of the homologous vertebrate gene, praxl, is not known. We describe the identification of the prox genes as members of a multigene family in vertebrates through the isolation of new members of the Prox gene family in zebrafish, Fugu rubripes, Tetraodon nigroviridis, mouse, and human. We examined the phylogeny of this new multigene family and we characterised the expression of these novel genes in zebrafish. Analysis of the expression of these genes identified the slow muscle as site of expression for prox 1 that did not overlap with the novel zebrafish Prox genes. Therefore, we studied the function ofprox 1 in the slow muscle using a combination of DNA, and morpholino injections. We demonstrate that prox 1 in not required for the specification of slow muscle as determined by the expression of markers of terminal differentiation. We also show that the medial lateral migration of the slow muscle is unaffected by the loss of prox 1. However, ectopic expression of prox 1 specifically in the fast muscle causes a defect in nuclear patterning. In normal development the fast muscle cells fuse early to form a multinucleate syncytium. The nuclei in this syncytium are normally evenly spaced. Ectopic expression of prox 1 resulted in the nuclei of the fast cells being positioned at the centre of the syncytium similarly to the situation observed in the mononucleate slow muscle. Furthermore loss of Prox 1 results in the disrupted patterning of the slow fibres, demonstrating a role for Prox 1 in the patterning of the slow muscle fibres. An understanding of the 3-dimensional (3D) pattern of gene expression can often lead to a better understanding of gene function. Optical projection tomography (OPT) is a new method for obtaining 3D data about an object. OPT generates a 3D digital model of a sample and allows it to be virtually sectioned, or rendered to produce a 3D image. OPT was developed for use on mouse embryos and had not been tested with zebrafish. We describe the difficulties of using OPT on samples as small as zebrafish embryos and the development of techniques to overcome these problems and allow its use in zebrafish

Identifying targets of the Sox domain protein Dichaete in the Drosophila CNS via targeted expression of dominant negative proteins.

BACKGROUND: Group B Sox domain transcription factors play important roles in metazoan central nervous system development. They are, however, difficult to study as mutations often have pleiotropic effects and other Sox family members can mask phenotypes due to functional compensation. In Drosophila melanogaster, the Sox gene Dichaete is dynamically expressed in the embryonic CNS, where it is known to have functional roles in neuroblasts and the ventral midline. In this study, we use inducible dominant negative proteins in combination with ChIP, immunohistochemistry and genome-wide expression profiling to further dissect the role of Dichaete in these two tissues. RESULTS: We generated two dominant negative Dichaete constructs, one lacking a DNA binding domain and the other fused to the Engrailed transcriptional repressor domain. We expressed these tissue-specifically in the midline and in neuroblasts using the UAS/GAL4 system, validating their use at the phenotypic level and with known target genes. Using ChIP and immunohistochemistry, we identified two new likely direct Dichaete target genes, commisureless in the midline and asense in the neuroectoderm. We performed genome-wide expression profiling in stage 8-9 embryos, identifying almost a thousand potential tissue-specific Dichaete targets, with half of these genes showing evidence of Dichaete binding in vivo. These include a number of genes with known roles in CNS development, including several components of the Notch, Wnt and EGFR signalling pathways. CONCLUSIONS: As well as identifying commisureless as a target, our data indicate that Dichaete helps establish its expression during early midline development but has less effect on its established later expression, highlighting Dichaete action on tissue specific enhancers. An analysis of the broader range of candidate Dichaete targets indicates that Dichaete plays diverse roles in CNS development, with the 500 or so Dichaete-bound putative targets including a number of transcription factors, signalling pathway components and terminal differentiation genes. In the early neurectoderm we implicate Dichaete in the lateral inhibition pathway and show that Dichaete acts to repress the proneural gene asense. Our analysis also reveals that dominant negatives cause off-target effects, highlighting the need to use other experimental data for validating findings from dominant negative studies.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Role of enteroendocrine cells in intestinal homeostasis and ageing.

The Intestine is a large and dynamic organ with a defined population of stem cells (ISCs) that are capable of giving rise to different differentiated cell types. The enteroendocrine cells (EEs) in the gut are responsible for producing different hormones which affect physiological and cellular processes locally and systemically throughout the body. In the intestine of the fruit fly (Drosophila melanogaster), EEs cumulatively produce 12 different prohormones that give rise to more than 20 hormone peptides. Despite their physiological importance, not much is known about the role that EEs and their products play in intestinal homeostasis during ageing in Drosophila. In the first part of my thesis, I investigated the mechanism by which the transcription factor Klumpfuss (Klu) determines the choice between enterocyte (EC) versus EE differentiation in the Drosophila intestine. We demonstrated that Klu acts in a cell-autonomous manner to restrict enteroblast (EB) cell fate to EC differentiation. Inhibition of klu expression by RNA interference resulted in excess EE differentiation. Ectopic expression of klu in ISCs reduces their proliferative capacity and blocked differentiation. Lastly, we showed that Klu acts down stream of Notch signaling in determining EC cell fate decision. In the second part of my thesis I employed a combination of bulk and single cell RNA sequencing (scRNA-seq) to investigate the changes in EE cells during ageing in both males and females. We observed significant changes in transcript-level for different EE hormones in our bulk RNA sequencing dataset. We examined the functional consequence of knocking down these hormones on ISC-proliferation and discovered that several EE-derived hormones play a role in promoting ISC-proliferation after infection. Moreover, we observed upregulation of pathways that are related to cell cycle and differentiation in old EEs, signifying changes in EE differentiation in the aged intestine. Analyzing our scRNA-seq dataset using Seurat algorithm and supervised clustering confirmed several of the changes from our bulk RNA-seq experiment, while expanding our knowledge on EE subtype change with age. We identified 4 major EE clusters in all conditions: NPF, AstA, AstC and EE progenitor cells, and observed increases in EE progenitor and AstA cell types in old female samples. Moreover, we observed a decrease of cells in the NPF cluster in old versus young female samples. This change was confirmed by Gal4 reporter line analysis for this hormone. Interestingly, the reduction of NPF-producing EEs in old flies happened in both male and female samples, but in different anatomical regions of the intestine. Finally, we showed that transcript of the transcription factor Escargot, which was thought to be restricted solely to the ISC and EB, is unexpectedly present in almost all EE clusters. With this study I have increased our understanding of ISC differentiation and cell fate determination in Drosophila intestine, and documented the changes that EE cells sustain during the ageing process. These results will be of crucial importance to better understand the role of EE cells in gut health and disease in an increasingly ageing population

Genetic and developmental mechanisms underlying the formation of the Drosophila compound eye

The compound eye of Drosophila melanogaster consists of individual subunits (“ommatidia”), each containing photoreceptors and support cells. These cells derive from an undifferentiated epithelium in the eye imaginal disc and their differentiation follows a highly stereotypic pattern. Sequential commitment of pluripotent cells to become specialized cells of the visual system serves as a unique model system to study basic mechanisms of tissue development. In the past years, many regulatory genes that govern the development of the compound eye have been identified and their mode of action genetically dissected. Transcription factor networks in combination with cell–cell signalling pathways regulate the development of the eye tissue in a precise temporal and spatial manner. Here, we review the recent advances on how a single-cell-layered epithelium is patterned to give rise to the compound eye. We discuss the molecular pathways controlling differentiation of individual photoreceptors, through which they acquire their functional specificity

Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems

The cnidarian-bilaterian ancestor possessed at least 56 homeoboxes: evidence from the starlet sea anemone, Nematostella vectensis

BACKGROUND: Homeodomain transcription factors are key components in the developmental toolkits of animals. While this gene superclass predates the evolutionary split between animals, plants, and fungi, many homeobox genes appear unique to animals. The origin of particular homeobox genes may, therefore, be associated with the evolution of particular animal traits. Here we report the first near-complete set of homeodomains from a basal (diploblastic) animal. RESULTS: Phylogenetic analyses were performed on 130 homeodomains from the sequenced genome of the sea anemone Nematostella vectensis along with 228 homeodomains from human and 97 homeodomains from Drosophila. The Nematostella homeodomains appear to be distributed among established homeodomain classes in the following fashion: 72 ANTP class; one HNF class; four LIM class; five POU class; 33 PRD class; five SINE class; and six TALE class. For four of the Nematostella homeodomains, there is disagreement between neighbor-joining and Bayesian trees regarding their class membership. A putative Nematostella CUT class gene is also identified. CONCLUSION: The homeodomain superclass underwent extensive radiations prior to the evolutionary split between Cnidaria and Bilateria. Fifty-six homeodomain families found in human and/or fruit fly are also found in Nematostella, though seventeen families shared by human and fly appear absent in Nematostella. Homeodomain loss is also apparent in the bilaterian taxa: eight homeodomain families shared by Drosophila and Nematostella appear absent from human (CG13424, EMXLX, HOMEOBRAIN, MSXLX, NK7, REPO, ROUGH, and UNC4), and six homeodomain families shared by human and Nematostella appear absent from fruit fly (ALX, DMBX, DUX, HNF, POU1, and VAX)