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Gao K, Donati A, Ainsworth J, Wu D, Terner ER, Perry MW. Deep conservation complemented by novelty and innovation in the insect eye ground plan. Proc Natl Acad Sci U S A 2025; 122:e2416562122. [PMID: 39793041 PMCID: PMC11725883 DOI: 10.1073/pnas.2416562122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Accepted: 11/09/2024] [Indexed: 01/12/2025] Open
Abstract
A spectacular diversity of forms and features allow species to thrive in different environments, yet some structures remain relatively unchanged. Insect compound eyes are easily recognizable despite dramatic differences in visual abilities across species. It is unknown whether distant insect species use similar or different mechanisms to pattern their eyes or what types of genetic changes produce diversity of form and function. We find that flies, mosquitos, butterflies, moths, beetles, wasps, honeybees, and crickets use homologous developmental programs to pattern their retinas. Transcription factor expression can be used to establish homology of different photoreceptor (PR) types across the insects: Prospero (Pros) for R7, Spalt (Sal) for R7+R8, and Defective proventriculus (Dve) for R1-6. Using gene knockout (CRISPR/Cas9) in houseflies, butterflies, and crickets and gene knockdown (RNAi) in beetles, we found that like Drosophila, EGFR and Sevenless (Sev) signaling pathways are required to recruit motion and color vision PRs, though Drosophila have a decreased reliance on Sev signaling relative to other insects. Despite morphological and physiological variation across species, retina development passes through a highly conserved phylotypic stage when the unit eyes (ommatidia) are first patterned. This patterning process likely represents an "insect eye ground plan" that is established by an ancient developmental program. We identify three types of developmental patterning modifications (ground plan modification, nonstochastic patterns, and specialized regions) that allow for the diversification of insect eyes. We suggest that developmental divergence after the ground plan is established is responsible for the exceptional diversity observed across insect visual systems.
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Affiliation(s)
- Ke Gao
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
| | - Antoine Donati
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
| | - Julia Ainsworth
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
| | - Di Wu
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
| | - Eleanor R. Terner
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
| | - Michael W. Perry
- Department of Cell & Developmental Biology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
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Aldrich JC, Vanderlinden LA, Jacobsen TL, Wood C, Saba LM, Britt SG. Genome-Wide Association Study and transcriptome analysis reveals a complex gene network that regulates opsin gene expression and cell fate determination in Drosophila R7 photoreceptor cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.05.606616. [PMID: 39149333 PMCID: PMC11326169 DOI: 10.1101/2024.08.05.606616] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 08/17/2024]
Abstract
Background An animal's ability to discriminate between differing wavelengths of light (i.e., color vision) is mediated, in part, by a subset of photoreceptor cells that express opsins with distinct absorption spectra. In Drosophila R7 photoreceptors, expression of the rhodopsin molecules, Rh3 or Rh4, is determined by a stochastic process mediated by the transcription factor spineless. The goal of this study was to identify additional factors that regulate R7 cell fate and opsin choice using a Genome Wide Association Study (GWAS) paired with transcriptome analysis via RNA-Seq. Results We examined Rh3 and Rh4 expression in a subset of fully-sequenced inbred strains from the Drosophila Genetic Reference Panel and performed a GWAS to identify 42 naturally-occurring polymorphisms-in proximity to 28 candidate genes-that significantly influence R7 opsin expression. Network analysis revealed multiple potential interactions between the associated candidate genes, spineless and its partners. GWAS candidates were further validated in a secondary RNAi screen which identified 12 lines that significantly reduce the proportion of Rh3 expressing R7 photoreceptors. Finally, using RNA-Seq, we demonstrated that all but four of the GWAS candidates are expressed in the pupal retina at a critical developmental time point and that five are among the 917 differentially expressed genes in sevenless mutants, which lack R7 cells. Conclusions Collectively, these results suggest that the relatively simple, binary cell fate decision underlying R7 opsin expression is modulated by a larger, more complex network of regulatory factors. Of particular interest are a subset of candidate genes with previously characterized neuronal functions including neurogenesis, neurodegeneration, photoreceptor development, axon growth and guidance, synaptogenesis, and synaptic function.
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Affiliation(s)
- John C. Aldrich
- Department of Neurology, Department of Ophthalmology, Dell Medical School; University of Texas at Austin, Austin, TX 78712
- Department of Psychology, University of Texas at Austin, Austin, TX 78712
| | - Lauren A. Vanderlinden
- Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
| | - Thomas L. Jacobsen
- Department of Neurology, Department of Ophthalmology, Dell Medical School; University of Texas at Austin, Austin, TX 78712
| | - Cheyret Wood
- Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
| | - Laura M. Saba
- Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
| | - Steven G. Britt
- Department of Neurology, Department of Ophthalmology, Dell Medical School; University of Texas at Austin, Austin, TX 78712
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Kitamata M, Otake Y, Kitagori H, Zhang X, Maki Y, Boku R, Takeuchi M, Nakagoshi H. Functional opsin patterning for Drosophila color vision is established through signaling pathways in adjacent object-detection neurons. Development 2024; 151:dev202388. [PMID: 38421315 PMCID: PMC10984275 DOI: 10.1242/dev.202388] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Accepted: 02/22/2024] [Indexed: 03/02/2024]
Abstract
Vision is mainly based on two different tasks, object detection and color discrimination, carried out by photoreceptor (PR) cells. The Drosophila compound eye consists of ∼800 ommatidia. Every ommatidium contains eight PR cells, six outer cells (R1-R6) and two inner cells (R7 and R8), by which object detection and color vision are achieved, respectively. Expression of opsin genes in R7 and R8 is highly coordinated through the instructive signal from R7 to R8, and two major ommatidial subtypes are distributed stochastically; pale type expresses Rh3/Rh5 and yellow type expresses Rh4/Rh6 in R7/R8. The homeodomain protein Defective proventriculus (Dve) is expressed in yellow-type R7 and in six outer PRs, and it is involved in Rh3 repression to specify the yellow-type R7. dve mutant eyes exhibited atypical coupling, Rh3/Rh6 and Rh4/Rh5, indicating that Dve activity is required for proper opsin coupling. Surprisingly, Dve activity in R1 is required for the instructive signal, whereas activity in R6 and R7 blocks the signal. Our results indicate that functional coupling of two different neurons is established through signaling pathways from adjacent neurons that are functionally different.
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Affiliation(s)
- Manabu Kitamata
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Yoshiaki Otake
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Hideaki Kitagori
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Xuanshuo Zhang
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Yusuke Maki
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Rika Boku
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Masato Takeuchi
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
| | - Hideki Nakagoshi
- Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
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Mishra AK, Sprecher SG. Eye Development in Drosophila : From Photoreceptor Specification to Terminal Differentiation. Neurogenetics 2023. [DOI: 10.1007/978-3-031-07793-7_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Valentino P, Erclik T. Spalt and disco define the dorsal-ventral neuroepithelial compartments of the developing Drosophila medulla. Genetics 2022; 222:iyac145. [PMID: 36135799 PMCID: PMC9630984 DOI: 10.1093/genetics/iyac145] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Accepted: 09/14/2022] [Indexed: 11/14/2022] Open
Abstract
Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center crescent are spatially patterned by the nonoverlapping expression of 3 transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the outer proliferation center and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the outer proliferation center also grow as distinct compartments, together with the fact that a subset of neuronal types is generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the outer proliferation center along the dorsal-ventral axis. Here, we identify the spalt (salm and salr) and disco (disco and disco-r) genes as the dorsal-ventral patterning transcription factors of the outer proliferation center. Spalt and Disco are differentially expressed in the dorsal and ventral outer proliferation center from the embryo through to the third instar larva, where they cross-repress each other to form a sharp dorsal-ventral boundary. We show that hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral outer proliferation center and its neuronal progeny. We further demonstrate that this dorsal-ventral patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third outer proliferation center patterning axis that may act to further diversify medulla fates.
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Affiliation(s)
- Priscilla Valentino
- Department of Biology, University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada
- Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 1A1, Canada
| | - Ted Erclik
- Department of Biology, University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada
- Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 1A1, Canada
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Bakovic V, Martin Cerezo ML, Höglund A, Fogelholm J, Henriksen R, Hargeby A, Wright D. The genomics of phenotypically differentiated Asellus aquaticus cave, surface stream and lake ecotypes. Mol Ecol 2021; 30:3530-3547. [PMID: 34002902 DOI: 10.1111/mec.15987] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2021] [Revised: 04/30/2021] [Accepted: 05/04/2021] [Indexed: 12/15/2022]
Abstract
Organisms well suited for the study of ecotype formation have wide distribution ranges, where they adapt to multiple drastically different habitats repeatedly over space and time. Here we study such ecotypes in a Crustacean model, Asellus aquaticus, a commonly occurring isopod found in freshwater habitats as diverse as streams, caves and lakes. Previous studies focusing on cave vs. surface ecotypes have attributed depigmentation, eye loss and prolonged antennae to several south European cave systems. Likewise, surveys across multiple Swedish lakes have identified the presence of dark-pigmented "reed" and light-pigmented "stonewort" ecotypes, which can be found within the same lake. In this study, we sequenced the first draft genome of A. aquaticus, and subsequently use this to map reads and call variants in surface stream, cave and two lake ecotypes. In addition, the draft genome was combined with a RADseq approach to perform a quantitative trait locus (QTL) mapping study using a laboratory bred F2 and F4 cave × surface intercross. We identified genomic regions associated with body pigmentation, antennae length and body size. Furthermore, we compared genome-wide differentiation between natural populations and found several genes potentially associated with these habitats. The assessment of the cave QTL regions in the light-dark comparison of lake populations suggests that the regions associated with cave adaptation are also involved with genomic differentiation in the lake ecotypes. These demonstrate how troglomorphic adaptations can be used as a model for related ecotype formation.
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Affiliation(s)
- Vid Bakovic
- IFM Biology, University of Linköping, Linköping, Sweden
| | | | | | | | - Rie Henriksen
- IFM Biology, University of Linköping, Linköping, Sweden
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Miller AC, Urban EA, Lyons EL, Herman TG, Johnston RJ. Interdependent regulation of stereotyped and stochastic photoreceptor fates in the fly eye. Dev Biol 2020; 471:89-96. [PMID: 33333066 PMCID: PMC7856283 DOI: 10.1016/j.ydbio.2020.12.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 12/07/2020] [Accepted: 12/09/2020] [Indexed: 12/14/2022]
Abstract
Diversification of neuronal subtypes often requires stochastic gene regulatory mechanisms. How stochastically expressed transcription factors interact with other regulators in gene networks to specify cell fates is poorly understood. The random mosaic of color-detecting R7 photoreceptor subtypes in Drosophila is controlled by the stochastic on/off expression of the transcription factor Spineless (Ss). In SsON R7s, Ss induces expression of Rhodopsin 4 (Rh4), whereas in SsOFF R7s, the absence of Ss allows expression of Rhodopsin 3 (Rh3). Here, we find that the transcription factor Runt, which is initially expressed in all R7s, is sufficient to promote stochastic Ss expression. Later, as R7s develop, Ss negatively feeds back onto Runt to prevent repression of Rh4 and ensure proper fate specification. Together, stereotyped and stochastic regulatory inputs are integrated into feedforward and feedback mechanisms to control cell fate.
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Affiliation(s)
- Adam C Miller
- Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403, USA
| | - Elizabeth A Urban
- Department of Biology, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218-2685, USA
| | - Eric L Lyons
- Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403, USA
| | - Tory G Herman
- Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403, USA.
| | - Robert J Johnston
- Department of Biology, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218-2685, USA.
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8
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Terzioğlu Kara E, Kiral FR, Öztürk Çolak A, Çelik A. Generation and characterization of inner photoreceptor-specific enhancer-trap lines using a novel piggyBac-Gal4 element in Drosophila. ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY 2020; 104:e21675. [PMID: 32285519 DOI: 10.1002/arch.21675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2020] [Revised: 03/17/2020] [Accepted: 03/23/2020] [Indexed: 06/11/2023]
Abstract
The Drosophila inner photoreceptors R7 and R8 are responsible for color vision and their differentiation starts at the third instar larval stage. Only a handful of genes with R7 or R8-cell-specific expression are known. We performed an enhancer-trap screen using a novel piggyBac transposable element, pBGay, carrying a Gal4 sequence under the control of the P promoter to identify novel genes expressed specifically in R7 or R8 cells. From this screen, three lines were analyzed in detail: piggyBacAC109 and piggyBacAC783 are expressed in R8 cells and piggyBacAC887 is expressed in R7 cells at the third instar larval stage and pupal stages. Molecular analysis showed that the piggyBac elements were inserted into the first intron of CG14160 and CG7985 genes and the second intron of unzipped. We show the expression pattern in the developing eye imaginal disc, pupal retina as well as the adult retina. The photoreceptor-specific expression of these genes is reported for the first time and we propose that these lines are useful tools for studying the development of the visual system.
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Affiliation(s)
- Ece Terzioğlu Kara
- Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bogazici University, Istanbul, Turkey
| | - Ferdi Rıdvan Kiral
- Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bogazici University, Istanbul, Turkey
- Division of Neurobiology, Institute for Biology, Free University Berlin, Berlin, Germany
| | - Arzu Öztürk Çolak
- Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bogazici University, Istanbul, Turkey
| | - Arzu Çelik
- Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bogazici University, Istanbul, Turkey
- Center for Life Sciences and Technologies, Bogazici University, Istanbul, Turkey
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9
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Chen YC, Desplan C. Gene regulatory networks during the development of the Drosophila visual system. Curr Top Dev Biol 2020; 139:89-125. [PMID: 32450970 DOI: 10.1016/bs.ctdb.2020.02.010] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The Drosophila visual system integrates input from 800 ommatidia and extracts different features in stereotypically connected optic ganglia. The development of the Drosophila visual system is controlled by gene regulatory networks that control the number of precursor cells, generate neuronal diversity by integrating spatial and temporal information, coordinate the timing of retinal and optic lobe cell differentiation, and determine distinct synaptic targets of each cell type. In this chapter, we describe the known gene regulatory networks involved in the development of the different parts of the visual system and explore general components in these gene networks. Finally, we discuss the advantages of the fly visual system as a model for gene regulatory network discovery in the era of single-cell transcriptomics.
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Affiliation(s)
- Yen-Chung Chen
- Department of Biology, New York University, New York, NY, United States
| | - Claude Desplan
- Department of Biology, New York University, New York, NY, United States.
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10
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Wang Y, Andongma AA, Dong Y, Chen Z, Xu P, Ren X, Krosch MN, Clarke AR, Niu C. Rh6 gene modulates the visual mechanism of host utilization in fruit fly Bactrocera minax. PEST MANAGEMENT SCIENCE 2019; 75:1621-1629. [PMID: 30471178 DOI: 10.1002/ps.5278] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/07/2018] [Revised: 11/05/2018] [Accepted: 11/15/2018] [Indexed: 06/09/2023]
Abstract
BACKGROUND Vision plays a critical role in host location and oviposition behavior for herbivorous insects. However, the molecular mechanisms underlying visual regulation in host recognition and oviposition site selection in insects remains unknown. The aim of this study was to explore the key visual genes that are linked to the host plant location of the fruit fly, Bactrocera minax. RESULTS Using a host specialist fruit fly, B. minax, which lays eggs only into immature green citrus fruit, we undertook behavioral, transcriptomic, and RNAi research to identify the molecular basis for host fruit color recognition. In laboratory and field assays we found that adults prefer green over other colors, and this preference is significantly increased in sexually mature over immature flies. Furthermore, we identified that the Rh6 gene, responsible for green spectral sensitivity, has elevated expression in mature flies over immature flies. RNAi suppression of Rh6 eliminated the preference for green, resulting in a significant decrease in the number of eggs laid by B. minax in green unripe citrus. CONCLUSION These results show that the Rh6 gene modulates the visual mechanism of host utilization in B. minax, providing a genetic basis for visual host location in a non-model insect herbivore. © 2018 Society of Chemical Industry.
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Affiliation(s)
- Yaohui Wang
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Awawing A Andongma
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Yongcheng Dong
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Zhenzhong Chen
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Penghui Xu
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Xueming Ren
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
| | - Matthew N Krosch
- School of Earth, Environmental and Biological Sciences, Faculty of Science and Technology, Queensland University of Technology (QUT), Brisbane, QLD, Australia
| | - Anthony R Clarke
- School of Earth, Environmental and Biological Sciences, Faculty of Science and Technology, Queensland University of Technology (QUT), Brisbane, QLD, Australia
| | - Changying Niu
- Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China
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Perry M, Konstantinides N, Pinto-Teixeira F, Desplan C. Generation and Evolution of Neural Cell Types and Circuits: Insights from the Drosophila Visual System. Annu Rev Genet 2017; 51:501-527. [PMID: 28961025 DOI: 10.1146/annurev-genet-120215-035312] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The Drosophila visual system has become a premier model for probing how neural diversity is generated during development. Recent work has provided deeper insight into the elaborate mechanisms that control the range of types and numbers of neurons produced, which neurons survive, and how they interact. These processes drive visual function and influence behavioral preferences. Other studies are beginning to provide insight into how neuronal diversity evolved in insects by adding new cell types and modifying neural circuits. Some of the most powerful comparisons have been those made to the Drosophila visual system, where a deeper understanding of molecular mechanisms allows for the generation of hypotheses about the evolution of neural anatomy and function. The evolution of new neural types contributes additional complexity to the brain and poses intriguing questions about how new neurons interact with existing circuitry. We explore how such individual changes in a variety of species might play a role over evolutionary timescales. Lessons learned from the fly visual system apply to other neural systems, including the fly central brain, where decisions are made and memories are stored.
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Affiliation(s)
- Michael Perry
- Department of Biology, New York University, New York, NY 10003, USA;
| | | | - Filipe Pinto-Teixeira
- Department of Biology, New York University, New York, NY 10003, USA; .,Center for Genomics and Systems Biology, New York University Abu Dhabi, Saadiyat Island, Abu Dhabi, United Arab Emirates
| | - Claude Desplan
- Department of Biology, New York University, New York, NY 10003, USA; .,Center for Genomics and Systems Biology, New York University Abu Dhabi, Saadiyat Island, Abu Dhabi, United Arab Emirates
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12
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Temporal profiling of photoreceptor lineage gene expression during murine retinal development. Gene Expr Patterns 2017; 23-24:32-44. [PMID: 28288836 DOI: 10.1016/j.gep.2017.03.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2016] [Revised: 02/03/2017] [Accepted: 03/07/2017] [Indexed: 02/08/2023]
Abstract
Rod and cone photoreceptors are photosensitive cells in the retina that convert light to electrical signals that are transmitted to visual processing centres in the brain. During development, cones and rods are generated from a common pool of multipotent retinal progenitor cells (RPCs) that also give rise to other retinal cell types. Cones and rods differentiate in two distinct waves, peaking in mid-embryogenesis and the early postnatal period, respectively. As RPCs transition from making cones to generating rods, there are changes in the expression profiles of genes involved in photoreceptor cell fate specification and differentiation. To better understand the temporal transition from cone to rod genesis, we assessed the timing of onset and offset of expression of a panel of 11 transcription factors and 7 non-transcription factors known to function in photoreceptor development, examining their expression between embryonic day (E) 12.5 and postnatal day (P) 60. Transcription factor expression in the photoreceptor layer was observed as early as E12.5, beginning with Crx, Otx2, Rorb, Neurod1 and Prdm1 expression, followed at E15.5 with the expression of Thrb, Neurog1, Sall3 and Rxrg expression, and at P0 by Nrl and Nr2e3 expression. Of the non-transcription factors, peanut agglutinin lectin staining and cone arrestin protein were observed as early as E15.5 in the developing outer nuclear layer, while transcripts for the cone opsins Opn1mw and Opn1sw and Recoverin protein were detected in photoreceptors by P0. In contrast, Opn1mw and Opn1sw protein were not observed in cones until P7, when rod-specific Gnat1 transcripts and rhodopsin protein were also detected. We have thus identified four transitory stages during murine retina photoreceptor differentiation marked by the period of onset of expression of new photoreceptor lineage genes. By characterizing these stages, we have clarified the dynamic nature of gene expression during the period when photoreceptor identities are progressively acquired during development.
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Yan J, Anderson C, Viets K, Tran S, Goldberg G, Small S, Johnston RJ. Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies. Development 2017; 144:844-855. [PMID: 28126841 DOI: 10.1242/dev.144030] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2016] [Accepted: 01/02/2017] [Indexed: 12/13/2022]
Abstract
How differential levels of gene expression are controlled in post-mitotic neurons is poorly understood. In the Drosophila retina, expression of the transcription factor Defective Proventriculus (Dve) at distinct cell type-specific levels is required for terminal differentiation of color- and motion-detecting photoreceptors. Here, we find that the activities of two cis-regulatory enhancers are coordinated to drive dve expression in the fly eye. Three transcription factors act on these enhancers to determine cell-type specificity. Negative autoregulation by Dve maintains expression from each enhancer at distinct homeostatic levels. One enhancer acts as an inducible backup ('dark' shadow enhancer) that is normally repressed but becomes active in the absence of the other enhancer. Thus, two enhancers integrate combinatorial transcription factor input, feedback and redundancy to generate cell type-specific levels of dve expression and stable photoreceptor fate. This regulatory logic may represent a general paradigm for how precise levels of gene expression are established and maintained in post-mitotic neurons.
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Affiliation(s)
- Jenny Yan
- Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2685, USA
| | - Caitlin Anderson
- Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2685, USA
| | - Kayla Viets
- Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2685, USA
| | - Sang Tran
- Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2685, USA
| | - Gregory Goldberg
- Center for Developmental Genetics, Department of Biology, New York University, 100 Washington Square East, New York, NY 10003-6688, USA
| | - Stephen Small
- Center for Developmental Genetics, Department of Biology, New York University, 100 Washington Square East, New York, NY 10003-6688, USA
| | - Robert J Johnston
- Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218-2685, USA
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Perry M, Kinoshita M, Saldi G, Huo L, Arikawa K, Desplan C. Molecular logic behind the three-way stochastic choices that expand butterfly colour vision. Nature 2016; 535:280-4. [PMID: 27383790 PMCID: PMC4988338 DOI: 10.1038/nature18616] [Citation(s) in RCA: 78] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Accepted: 05/31/2016] [Indexed: 01/08/2023]
Abstract
Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. We show that the Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (Spineless(ON)) or an ultraviolet (UV)-sensitive (Spineless(OFF)) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 (refs 5, 6) leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.
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Affiliation(s)
- Michael Perry
- Department of Biology, New York University, New York, New York 10003, USA
| | - Michiyo Kinoshita
- Laboratory of Neuroethology, Department of Evolutionary Studies of Biosystems, SOKENDAI, The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0115, Japan
| | - Giuseppe Saldi
- New York University Abu Dhabi, Saadiyat Island, Abu Dhabi, United Arab Emirates
| | - Lucy Huo
- Department of Biology, New York University, New York, New York 10003, USA
| | - Kentaro Arikawa
- Laboratory of Neuroethology, Department of Evolutionary Studies of Biosystems, SOKENDAI, The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0115, Japan
| | - Claude Desplan
- Department of Biology, New York University, New York, New York 10003, USA.,New York University Abu Dhabi, Saadiyat Island, Abu Dhabi, United Arab Emirates
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15
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Abstract
It has been nearly 15 years since the suggestion that synaptically released Zn2+ might contribute to excitotoxic brain injury after seizures, stroke, and brain trauma. In the original “zinc-translocation” model, it was proposed that synaptically released Zn2+ ions penetrated postsynaptic neurons, causing injury. According to the model, chelating zinc in the cleft was predicted to be neuroprotective. This proved to be true: zinc chelators have proved to be remarkably potent at reducing excitotoxic neuronal injury in many paradigms. Promising new zinc-based therapies for stroke, head trauma, and epileptic brain injury are under development. However, new evidence suggests that the original translocation model was incomplete. As many as three sources of toxic zinc ions may contribute to excitotoxicity: presynaptic vesicles, postsynaptic zincsequestering proteins, and (more speculatively) mitochondrial pools. The authors present a new model of zinc currents and zinc toxicity that offers expanded opportunities for zinc-selective therapeutic chelation interventions.
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16
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Chouhan AK, Guo C, Hsieh YC, Ye H, Senturk M, Zuo Z, Li Y, Chatterjee S, Botas J, Jackson GR, Bellen HJ, Shulman JM. Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease. Acta Neuropathol Commun 2016; 4:62. [PMID: 27338814 PMCID: PMC4918017 DOI: 10.1186/s40478-016-0333-4] [Citation(s) in RCA: 58] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 06/07/2016] [Indexed: 02/04/2023] Open
Abstract
Common neurodegenerative proteinopathies, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by the misfolding and aggregation of toxic protein species, including the amyloid beta (Aß) peptide, microtubule-associated protein Tau (Tau), and alpha-synuclein (αSyn) protein. These factors also show toxicity in Drosophila; however, potential limitations of prior studies include poor discrimination between effects on the adult versus developing nervous system and neuronal versus glial cell types. In addition, variable expression paradigms and outcomes hinder systematic comparison of toxicity profiles. Using standardized conditions and medium-throughput assays, we express human Tau, Aß or αSyn selectively in neurons of the adult Drosophila retina and monitor age-dependent changes in both structure and function, based on tissue histology and recordings of the electroretinogram (ERG), respectively. We find that each protein causes a unique profile of neurodegenerative pathology, demonstrating distinct and separable impacts on neuronal death and dysfunction. Strikingly, expression of Tau leads to progressive loss of ERG responses whereas retinal architecture and neuronal numbers are largely preserved. By contrast, Aß induces modest, age-dependent neuronal loss without degrading the retinal ERG. αSyn expression, using a codon-optimized transgene, is characterized by marked retinal vacuolar change, progressive photoreceptor cell death, and delayed-onset but modest ERG changes. Lastly, to address potential mechanisms, we perform transmission electron microscopy (TEM) to reveal potential degenerative changes at the ultrastructural level. Surprisingly, Tau and αSyn each cause prominent but distinct synaptotoxic profiles, including disorganization or enlargement of photoreceptor terminals, respectively. Our findings highlight variable and dynamic properties of neurodegeneration triggered by these disease-relevant proteins in vivo, and suggest that Drosophila may be useful for revealing determinants of neuronal dysfunction that precede cell loss, including synaptic changes, in the adult nervous system.
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17
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Functional genomics identifies regulators of the phototransduction machinery in the Drosophila larval eye and adult ocelli. Dev Biol 2016; 410:164-177. [PMID: 26769100 DOI: 10.1016/j.ydbio.2015.12.026] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2015] [Revised: 12/22/2015] [Accepted: 12/22/2015] [Indexed: 11/21/2022]
Abstract
Sensory perception of light is mediated by specialized Photoreceptor neurons (PRs) in the eye. During development all PRs are genetically determined to express a specific Rhodopsin (Rh) gene and genes mediating a functional phototransduction pathway. While the genetic and molecular mechanisms of PR development is well described in the adult compound eye, it remains unclear how the expression of Rhodopsins and the phototransduction cascade is regulated in other visual organs in Drosophila, such as the larval eye and adult ocelli. Using transcriptome analysis of larval PR-subtypes and ocellar PRs we identify and study new regulators required during PR differentiation or necessary for the expression of specific signaling molecules of the functional phototransduction pathway. We found that the transcription factor Krüppel (Kr) is enriched in the larval eye and controls PR differentiation by promoting Rh5 and Rh6 expression. We also identified Camta, Lola, Dve and Hazy as key genes acting during ocellar PR differentiation. Further we show that these transcriptional regulators control gene expression of the phototransduction cascade in both larval eye and adult ocelli. Our results show that PR cell type-specific transcriptome profiling is a powerful tool to identify key transcriptional regulators involved during several aspects of PR development and differentiation. Our findings greatly contribute to the understanding of how combinatorial action of key transcriptional regulators control PR development and the regulation of a functional phototransduction pathway in both larval eye and adult ocelli.
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18
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Wernet MF, Perry MW, Desplan C. The evolutionary diversity of insect retinal mosaics: common design principles and emerging molecular logic. Trends Genet 2015; 31:316-28. [PMID: 26025917 PMCID: PMC4458154 DOI: 10.1016/j.tig.2015.04.006] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2015] [Revised: 04/13/2015] [Accepted: 04/14/2015] [Indexed: 12/21/2022]
Abstract
Independent evolution has resulted in a vast diversity of eyes. Despite the lack of a common Bauplan or ancestral structure, similar developmental strategies are used. For instance, different classes of photoreceptor cells (PRs) are distributed stochastically and/or localized in different regions of the retina. Here, we focus on recent progress made towards understanding the molecular principles behind patterning retinal mosaics of insects, one of the most diverse groups of animals adapted to life on land, in the air, under water, or on the water surface. Morphological, physiological, and behavioral studies from many species provide detailed descriptions of the vast variation in retinal design and function. By integrating this knowledge with recent progress in the characterization of insect Rhodopsins as well as insight from the model organism Drosophila melanogaster, we seek to identify the molecular logic behind the adaptation of retinal mosaics to the habitat and way of life of an animal.
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Affiliation(s)
- Mathias F Wernet
- New York University Abu Dhabi, Abu Dhabi, 129188 Saadiyat Island, United Arab Emirates
| | - Michael W Perry
- Department of Biology, New York University, New York, NY 10003, USA
| | - Claude Desplan
- New York University Abu Dhabi, Abu Dhabi, 129188 Saadiyat Island, United Arab Emirates; Department of Biology, New York University, New York, NY 10003, USA.
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19
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Wernet MF, Desplan C. Homothorax and Extradenticle alter the transcription factor network in Drosophila ommatidia at the dorsal rim of the retina. Development 2014; 141:918-28. [PMID: 24496628 DOI: 10.1242/dev.103127] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
A narrow band of ommatidia in the dorsal periphery of the Drosophila retina called the dorsal rim area (DRA) act as detectors for polarized light. The transcription factor Homothorax (Hth) is expressed in DRA inner photoreceptors R7 and R8 and is both necessary and sufficient to induce the DRA fate, including specialized morphology and unique Rhodopsin expression. Hth expression is the result of Wingless (Wg) pathway activity at the eye margins and restriction to the dorsal eye by the selector genes of the Iroquois complex (Iro-C). However, how the DRA is limited to exactly one or two ommatidial rows is not known. Although several factors regulating the Drosophila retinal mosaic are expressed in DRA ommatidia, the role of Hth in this transcriptional network is uncharacterized. Here we show that Hth functions together with its co-factor Extradenticle (Exd) to repress the R8-specific factor Senseless (Sens) in DRA R8 cells, allowing expression of an ultraviolet-sensitive R7 Rhodopsin (Rh3). Furthermore, Hth/Exd act in concert with the transcriptional activators Orthodenticle (Otd) and Spalt (Sal), to activate expression of Rh3 in the DRA. The resulting monochromatic coupling of Rh3 between R7 and R8 in DRA ommatidia is important for comparing celestial e-vector orientation rather than wavelengths. Finally, we show that Hth expression expands to many ommatidial rows in regulatory mutants of optomotorblind (omb), a transcription factor transducing Wg signaling at the dorsal and ventral eye poles. Therefore, locally restricted recruitment of the DRA-specific factor Hth alters the transcriptional network that regulates Rhodopsin expression across ommatidia.
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Affiliation(s)
- Mathias F Wernet
- Center for Developmental Genetics, Department of Biology, New York University, 100 Washington Place, New York, NY 10003, USA
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20
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Fan X, Huang Q, Ye X, Lin Y, Chen Y, Lin X, Qu J. Drosophila USP5 controls the activation of apoptosis and the Jun N-terminal kinase pathway during eye development. PLoS One 2014; 9:e92250. [PMID: 24643212 PMCID: PMC3958489 DOI: 10.1371/journal.pone.0092250] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2013] [Accepted: 02/20/2014] [Indexed: 11/18/2022] Open
Abstract
The Jun N-terminal kinase pathway plays an important role in inducing programmed cell death (apoptosis) and is activated in a variety of contexts. The deubiquitinating enzymes (DUBs) are proteases regulating the protein stability by ubiquitin-proteasome system. Here, for the first time, we report the phenotypes observed during eye development that are induced by deleting Drosophila USP5 gene, which encodes one of the USP subfamily of DUBs. usp5 mutants displayed defects in photoreceptor differentiation. Using genetic epistasis analysis and molecular markers, we show that most of these phenotypes are caused by the activation of apoptosis and JNK pathway. These data may provide a mechanistic model for understanding the mammalian usp5 gene.
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Affiliation(s)
- Xiaolan Fan
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Qinzhu Huang
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Xiaolei Ye
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yi Lin
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yuting Chen
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Xinhua Lin
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Jia Qu
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
- * E-mail:
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21
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Genetic dissection of photoreceptor subtype specification by the Drosophila melanogaster zinc finger proteins elbow and no ocelli. PLoS Genet 2014; 10:e1004210. [PMID: 24625735 PMCID: PMC3953069 DOI: 10.1371/journal.pgen.1004210] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2013] [Accepted: 01/15/2014] [Indexed: 12/28/2022] Open
Abstract
The elbow/no ocelli (elb/noc) complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. Here we show that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the 'dorsal rim area' (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. Our data suggest that Elb/Noc genetically interact with Hth, and we identify two conserved domains crucial for this function. Furthermore, we show that Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. We propose that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease.
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22
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Johnston RJ, Desplan C. Interchromosomal communication coordinates intrinsically stochastic expression between alleles. Science 2014; 343:661-5. [PMID: 24503853 DOI: 10.1126/science.1243039] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Sensory systems use stochastic mechanisms to diversify neuronal subtypes. In the Drosophila eye, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) determines a random binary subtype choice in R7 photoreceptors. Here, we show that a stochastic, cell-autonomous decision to express ss is made intrinsically by each ss locus. Stochastic on or off expression of each ss allele is determined by combinatorial inputs from one enhancer and two silencers acting at long range. However, the two ss alleles also average their frequency of expression through up-regulatory and down-regulatory interallelic cross-talk. This inter- or intrachromosomal long-range regulation does not require endogenous ss chromosomal positioning or pairing. Therefore, although individual ss alleles make independent stochastic choices, interchromosomal communication coordinates expression state between alleles, ensuring that they are both expressed in the same random subset of R7s.
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Affiliation(s)
- Robert J Johnston
- Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA
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23
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Mishra AK, Tsachaki M, Rister J, Ng J, Celik A, Sprecher SG. Binary cell fate decisions and fate transformation in the Drosophila larval eye. PLoS Genet 2013; 9:e1004027. [PMID: 24385925 PMCID: PMC3873242 DOI: 10.1371/journal.pgen.1004027] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2013] [Accepted: 10/30/2013] [Indexed: 11/18/2022] Open
Abstract
The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.
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Affiliation(s)
- Abhishek Kumar Mishra
- Institute of Cell and Developmental Biology, Department of Biology, University of Fribourg, Fribourg, Switzerland
| | - Maria Tsachaki
- Institute of Cell and Developmental Biology, Department of Biology, University of Fribourg, Fribourg, Switzerland
| | - Jens Rister
- Center for Developmental Genetics, Department of Biology, New York University, New York, New York, United States of America
| | - June Ng
- Center for Developmental Genetics, Department of Biology, New York University, New York, New York, United States of America
| | - Arzu Celik
- Department of Molecular Biology and Genetics, Bogazici University, Bebek, Istanbul, Turkey
| | - Simon G. Sprecher
- Institute of Cell and Developmental Biology, Department of Biology, University of Fribourg, Fribourg, Switzerland
- * E-mail:
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24
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Coelho DS, Cairrão F, Zeng X, Pires E, Coelho AV, Ron D, Ryoo HD, Domingos PM. Xbp1-independent Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila. Cell Rep 2013; 5:791-801. [PMID: 24183663 PMCID: PMC3858604 DOI: 10.1016/j.celrep.2013.09.046] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2012] [Revised: 08/28/2013] [Accepted: 09/30/2013] [Indexed: 10/26/2022] Open
Abstract
The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the fatty acid transport protein (fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.
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Affiliation(s)
- Dina S. Coelho
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Fatima Cairrão
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Xiaomei Zeng
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, USA
| | - Elisabete Pires
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Ana V. Coelho
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - David Ron
- University of Cambridge, Metabolic Research Laboratory and NIHR Cambridge Biomedical Research Centre, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK
| | - Hyung Don Ryoo
- Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, USA
| | - Pedro M. Domingos
- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
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25
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Spletter ML, Schnorrer F. Transcriptional regulation and alternative splicing cooperate in muscle fiber-type specification in flies and mammals. Exp Cell Res 2013; 321:90-8. [PMID: 24145055 PMCID: PMC4040393 DOI: 10.1016/j.yexcr.2013.10.007] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2013] [Revised: 10/06/2013] [Accepted: 10/09/2013] [Indexed: 11/21/2022]
Abstract
Muscles coordinate body movements throughout the animal kingdom. Each skeletal muscle is built of large, multi-nucleated cells, called myofibers, which are classified into several functionally distinct types. The typical fiber-type composition of each muscle arises during development, and in mammals is extensively adjusted in response to postnatal exercise. Understanding how functionally distinct muscle fiber-types arise is important for unraveling the molecular basis of diseases from cardiomyopathies to muscular dystrophies. In this review, we focus on recent advances in Drosophila and mammals in understanding how muscle fiber-type specification is controlled by the regulation of transcription and alternative splicing. We illustrate the cooperation of general myogenic transcription factors with muscle fiber-type specific transcriptional regulators as a basic principle for fiber-type specification, which is conserved from flies to mammals. We also examine how regulated alternative splicing of sarcomeric proteins in both flies and mammals can directly instruct the physiological and biophysical differences between fiber-types. Thus, research in Drosophila can provide important mechanistic insight into muscle fiber specification, which is relevant to homologous processes in mammals and to the pathology of muscle diseases.
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Affiliation(s)
- Maria L Spletter
- Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
| | - Frank Schnorrer
- Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
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26
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Transcriptional regulation of tissue organization and cell morphogenesis: the fly retina as a case study. Dev Biol 2013; 385:168-78. [PMID: 24099926 DOI: 10.1016/j.ydbio.2013.09.031] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2013] [Revised: 09/27/2013] [Accepted: 09/29/2013] [Indexed: 12/11/2022]
Abstract
Understanding how a functional organ can be produced from a small group of cells remains an outstanding question in cell and developmental biology. The developing compound eye of Drosophila has long been a model of choice for addressing this question by dissecting the cellular, genetic and molecular pathways that govern cell specification, differentiation, and multicellular patterning during organogenesis. In this review, the author focussed on cell and tissue morphogenesis during fly retinal development, including the regulated changes in cell shape and cell packing that ultimately determine the shape and architecture of the compound eye. In particular, the author reviewed recent studies that highlight the prominent roles of transcriptional and hormonal controls that orchestrate the cell shape changes, cell-cell junction remodeling and polarized membrane growth that underlie photoreceptor morphogenesis and retinal patterning.
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27
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Dourlen P, Levet C, Mejat A, Gambis A, Mollereau B. The Tomato/GFP-FLP/FRT method for live imaging of mosaic adult Drosophila photoreceptor cells. J Vis Exp 2013:e50610. [PMID: 24084155 PMCID: PMC3923918 DOI: 10.3791/50610] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.
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Affiliation(s)
- Pierre Dourlen
- Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon
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28
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The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes. Dev Biol 2013; 381:482-90. [PMID: 23850772 DOI: 10.1016/j.ydbio.2013.07.001] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2013] [Revised: 06/15/2013] [Accepted: 07/03/2013] [Indexed: 01/27/2023]
Abstract
Signaling pathways are often re-used during development in surprisingly different ways. The Hippo tumor suppressor pathway is best understood for its role in the control of growth. The pathway is also used in a very different context, in the Drosophila eye for the robust specification of R8 photoreceptor neuron subtypes, which complete their terminal differentiation by expressing light-sensing Rhodopsin (Rh) proteins. A double negative feedback loop between the Warts kinase of the Hippo pathway and the PH-domain growth regulator Melted regulates the choice between 'pale' R8 (pR8) fate defined by Rh5 expression and 'yellow' R8 (yR8) fate characterized by Rh6 expression. Here, we show that the gene encoding the homolog of human Nuclear respiratory factor 1, erect wing (ewg), is autonomously required to inhibit warts expression and to promote melted expression to specify pR8 subtype fate and induce Rh5. ewg mutants express Rh6 in most R8s due to ectopic warts expression. Further, ewg is continuously required to maintain repression of Rh6 in pR8s in aging flies. Our work shows that Ewg is a critical factor for the stable down-regulation of Hippo pathway activity to determine neuronal subtype fates. Neural-enriched factors, such as Ewg, may generally contribute to the contextual re-use of signaling pathways in post-mitotic neurons.
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Johnston RJ. Lessons about terminal differentiation from the specification of color-detecting photoreceptors in the Drosophila retina. Ann N Y Acad Sci 2013; 1293:33-44. [PMID: 23782311 DOI: 10.1111/nyas.12178] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Metazoans require highly diverse collections of cell types to sense, interpret, and react to the environment. Developmental programs incorporate deterministic and stochastic strategies in different contexts or different combinations to establish this multitude of cell fates. Precise genetic dissection of the processes controlling terminal photoreceptor differentiation in the Drosophila retina has revealed complex regulatory mechanisms required to generate differences in gene expression and cell fate. In this review, I discuss how a gene regulatory network interprets stochastic and regional inputs to determine the specification of color-detecting photoreceptor subtypes in the Drosophila retina. These combinatorial gene regulatory mechanisms will likely be broadly applicable to nervous system development and cell fate specification in general.
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Affiliation(s)
- Robert J Johnston
- Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
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30
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Rister J, Desplan C, Vasiliauskas D. Establishing and maintaining gene expression patterns: insights from sensory receptor patterning. Development 2013; 140:493-503. [PMID: 23293281 DOI: 10.1242/dev.079095] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
In visual and olfactory sensory systems with high discriminatory power, each sensory neuron typically expresses one, or very few, sensory receptor genes, excluding all others. Recent studies have provided insights into the mechanisms that generate and maintain sensory receptor expression patterns. Here, we review how this is achieved in the fly retina and compare it with the mechanisms controlling sensory receptor expression patterns in the mouse retina and in the mouse and fly olfactory systems.
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Affiliation(s)
- Jens Rister
- Department of Biology, New York University, 1009 Silver Center, 100 Washington Square East, New York, NY 10003-6688, USA
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31
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Treisman JE. Retinal differentiation in Drosophila. WILEY INTERDISCIPLINARY REVIEWS-DEVELOPMENTAL BIOLOGY 2012; 2:545-57. [PMID: 24014422 DOI: 10.1002/wdev.100] [Citation(s) in RCA: 97] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Drosophila eye development has been extensively studied, due to the ease of genetic screens for mutations disrupting this process. The eye imaginal disc is specified during embryonic and larval development by the Pax6 homolog Eyeless and a network of downstream transcription factors. Expression of these factors is regulated by signaling molecules and also indirectly by growth of the eye disc. Differentiation of photoreceptor clusters initiates in the third larval instar at the posterior of the eye disc and progresses anteriorly, driven by the secreted protein Hedgehog. Within each cluster, the combined activities of Hedgehog signaling and Notch-mediated lateral inhibition induce and refine the expression of the transcription factor Atonal, which specifies the founding R8 photoreceptor of each ommatidium. Seven additional photoreceptors, followed by cone and pigment cells, are successively recruited by the signaling molecules Spitz, Delta, and Bride of sevenless. Combinations of these signals and of intrinsic transcription factors give each ommatidial cell its specific identity. During the pupal stages, rhodopsins are expressed, and the photoreceptors and accessory cells take on their final positions and morphologies to form the adult retina. Over the past few decades, the genetic analysis of this small number of cell types arranged in a repetitive structure has allowed a remarkably detailed understanding of the basic mechanisms controlling cell differentiation and morphological rearrangement.
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Affiliation(s)
- Jessica E Treisman
- Department of Cell Biology and Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine, New York, NY, USA.
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32
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Organista MF, De Celis JF. The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway. Biol Open 2012; 2:37-48. [PMID: 23336075 PMCID: PMC3545267 DOI: 10.1242/bio.20123038] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2012] [Accepted: 09/19/2012] [Indexed: 01/25/2023] Open
Abstract
The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade.
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Affiliation(s)
- María F Organista
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco , Madrid 28049 , Spain
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33
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Abstract
PURPOSE OF REVIEW Stem cell gene SALL4 has been well characterized for its essential role in developmental events as well as embryonic stem cell pluripotency maintenance. Several current reports now shed new light on its functions in regulating hematopoietic cell self-renewal and differentiation. In this review we attempt to summarize SALL4 roles for normal hematopoiesis, and how the knowledge obtained can be used to develop advanced cell therapies. RECENT FINDINGS SALL4 may act as a critical controller to regulate the fate of hematopoietic cells. In normal bone marrow, SALL4 is selectively expressed in primitive hematopoietic precursors and rapidly downregulated following differentiation. Of particular interest, SALL4 isoforms are able to stimulate large scale ex-vivo expansion of hematopoietic stem/progenitor cells (HSCs/HPCs). The SALL4 expanded HSCs/HPCs retain multilineage repopulation and long-term engraftment activities, which are clinically meaningful. The stem cell self-renewal mediated by SALL4 is linked to epigenetic machinery. SUMMARY The emerging knowledge about how SALL4 regulates HSC behavior may be used in the near future to develop advanced cell therapies, for example, through large-scale stem cell expansion ex vivo.
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Yang XX, Sun JZ, Li FX, Wu YS, Du HY, Zhu W, Li XH, Li M. Aberrant methylation and downregulation of sall3 in human hepatocellular carcinoma. World J Gastroenterol 2012; 18:2719-26. [PMID: 22690083 PMCID: PMC3370011 DOI: 10.3748/wjg.v18.i21.2719] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2011] [Revised: 10/27/2011] [Accepted: 04/28/2012] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigated whether sall3 transcription was regulated by promoter CpG island hypermethylation in hepatocellular carcinoma (HCC).
METHODS: The cell lines Huh7, HepG2, SK-HEP1, SMMC7721, Bel7402, QGY7703 and a cohort of 38 HCC tissue specimens and corresponding nontumorous tissues were subjected to analysis for sall3 promoter CpG island methylation and mRNA transcription. sall3 promoter CpG island methylation levels were determined using the MassARRAY platform and mRNA transcription levels of the gene were detected by quantitative real-time polymerase chain reaction.
RESULTS: The levels of sall3 mRNA were decreased by more than twofold in 33 of 38 tumor tissues compared to adjacent noncancerous tissues. Among these 33 tumor tissues with lower levels of sall3 mRNA, 24 showed higher levels of methylation. Based on these results, we hypothesized that the decrease in sall3 mRNA transcription level was likely due to promoter CpG island hypermethylation. Changes in sall3 mRNA transcription and promoter CpG island methylation were determined in the above six cell lines after treatment with 0, 0.1, 0.5 and 2.5 μmol 5-aza-2-deoxycytidine, a demethylating agent. Promoter CpG island methylation levels decreased in a dose-dependent manner in all six cell lines, while the mRNA transcription level increased dose-dependently in Huh7, HepG2, SK-HEP1 and SMMC7721 cells and irregularly in Bel7402 and QGY7703 cells.
CONCLUSION: These results indicated that promoter CpG island hypermethylation contributes to the downregulation of sall3 mRNA transcription in HCC.
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35
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Quan XJ, Ramaekers A, Hassan BA. Transcriptional control of cell fate specification: lessons from the fly retina. Curr Top Dev Biol 2012; 98:259-76. [PMID: 22305166 DOI: 10.1016/b978-0-12-386499-4.00010-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/25/2023]
Abstract
It is now widely recognized that as cells of developing tissues transition through successive states of decreasing pluripotency into a state of terminal differentiation, they undergo significant changes in their gene expression profiles. Interestingly, these successive states of increasing differentiation are marked by the spatially and temporally restricted expression of sets of transcription factors. Each wave of transcription factors not only signals the arrival of a given stage in cellular differentiation, but it is also necessary for the activation of the next set of transcription factors, creating the appearance of a smooth, directed, and deterministic genetic program of cellular differentiation. Until recently, however, it was largely unknown which genes, besides each other, these transcription factors were activating. Thus, the molecular definition of any given step of differentiation, and how it gave rise to the following step remained unclear. Recent advances in transcriptomics, bioinformatics, and molecular genetics resulted in the identification of numerous transcription factor target genes (TGs). These advances have opened the door to using similar approaches in developmental biology to understand what the transcriptional cascades of cellular differentiation might be. Using the development of the Drosophila eye as a model system, we discuss the role of transcription factors and their TGs in cell fate specification and terminal differentiation.
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Affiliation(s)
- Xiao-jiang Quan
- Laboratory of Neurogenetics, VIB Center for the Biology of Disease, VIB, KU Leuven School of Medicine, Leuven, Belgium
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36
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Abstract
Color vision is found in many invertebrate and vertebrate species. It is the ability to discriminate objects based on the wavelength of emitted light independent of intensity. As it requires the comparison of at least two photoreceptor types with different spectral sensitivities, this process is often mediated by a mosaic made of several photoreceptor types. In this review, we summarize the current knowledge about the formation of retinal mosaics and the regulation of photopigment (opsin) expression in the fly, mouse, and human retina. Despite distinct evolutionary origins, as well as major differences in morphology and phototransduction machineries, there are significant similarities in the stepwise cell-fate decisions that lead from progenitor cells to terminally differentiated photoreceptors that express a particular opsin. Common themes include (i) the use of binary transcriptional switches that distinguish classes of photoreceptors, (ii) the use of gradients of signaling molecules for regional specializations, (iii) stochastic choices that pattern the retina, and (iv) the use of permissive factors with multiple roles in different photoreceptor types.
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Affiliation(s)
- Jens Rister
- Department of Biology, Center for Developmental Genetics, New York University, USA
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37
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Schönbauer C, Distler J, Jährling N, Radolf M, Dodt HU, Frasch M, Schnorrer F. Spalt mediates an evolutionarily conserved switch to fibrillar muscle fate in insects. Nature 2011; 479:406-9. [PMID: 22094701 DOI: 10.1038/nature10559] [Citation(s) in RCA: 79] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2011] [Accepted: 09/13/2011] [Indexed: 11/09/2022]
Abstract
Flying insects oscillate their wings at high frequencies of up to 1,000 Hz and produce large mechanical forces of 80 W per kilogram of muscle. They utilize a pair of perpendicularly oriented indirect flight muscles that contain fibrillar, stretch-activated myofibres. In contrast, all other, more slowly contracting, insect body muscles have a tubular muscle morphology. Here we identify the transcription factor Spalt major (Salm) as a master regulator of fibrillar flight muscle fate in Drosophila. salm is necessary and sufficient to induce fibrillar muscle fate. salm switches the entire transcriptional program from tubular to fibrillar fate by regulating the expression and splicing of key sarcomeric components specific to each muscle type. Spalt function is conserved in insects evolutionarily separated by 280 million years. We propose that Spalt proteins switch myofibres from tubular to fibrillar fate during development, a function potentially conserved in the vertebrate heart--a stretch-activated muscle sharing features with insect flight muscle.
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Affiliation(s)
- Cornelia Schönbauer
- Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
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38
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Neumüller RA, Richter C, Fischer A, Novatchkova M, Neumüller KG, Knoblich JA. Genome-wide analysis of self-renewal in Drosophila neural stem cells by transgenic RNAi. Cell Stem Cell 2011; 8:580-93. [PMID: 21549331 PMCID: PMC3093620 DOI: 10.1016/j.stem.2011.02.022] [Citation(s) in RCA: 208] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2009] [Revised: 11/01/2010] [Accepted: 02/16/2011] [Indexed: 01/14/2023]
Abstract
The balance between stem cell self-renewal and differentiation is precisely controlled to ensure tissue homeostasis and prevent tumorigenesis. Here we use genome-wide transgenic RNAi to identify 620 genes potentially involved in controlling this balance in Drosophila neuroblasts. We quantify all phenotypes and derive measurements for proliferation, lineage, cell size, and cell shape. We identify a set of transcriptional regulators essential for self-renewal and use hierarchical clustering and integration with interaction data to create functional networks for the control of neuroblast self-renewal and differentiation. Our data identify key roles for the chromatin remodeling Brm complex, the spliceosome, and the TRiC/CCT-complex and show that the alternatively spliced transcription factor Lola and the transcriptional elongation factors Ssrp and Barc control self-renewal in neuroblast lineages. As our data are strongly enriched for genes highly expressed in murine neural stem cells, they are likely to provide valuable insights into mammalian stem cell biology as well.
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Affiliation(s)
- Ralph A Neumüller
- Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
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39
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Tsachaki M, Sprecher SG. Genetic and developmental mechanisms underlying the formation of theDrosophilacompound eye. Dev Dyn 2011; 241:40-56. [DOI: 10.1002/dvdy.22738] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/24/2011] [Indexed: 01/15/2023] Open
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40
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de Melo J, Peng GH, Chen S, Blackshaw S. The Spalt family transcription factor Sall3 regulates the development of cone photoreceptors and retinal horizontal interneurons. Development 2011; 138:2325-36. [PMID: 21558380 PMCID: PMC3091496 DOI: 10.1242/dev.061846] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The mammalian retina is a tractable model system for analyzing transcriptional networks that guide neural development. Spalt family zinc-finger transcription factors play a crucial role in photoreceptor specification in Drosophila, but their role in mammalian retinal development has not been investigated. In this study, we show that that the spalt homolog Sall3 is prominently expressed in developing cone photoreceptors and horizontal interneurons of the mouse retina and in a subset of cone bipolar cells. We find that Sall3 is both necessary and sufficient to activate the expression of multiple cone-specific genes, and that Sall3 protein is selectively bound to the promoter regions of these genes. Notably, Sall3 shows more prominent expression in short wavelength-sensitive cones than in medium wavelength-sensitive cones, and that Sall3 selectively activates expression of the short but not the medium wavelength-sensitive cone opsin gene. We further observe that Sall3 regulates the differentiation of horizontal interneurons, which form direct synaptic contacts with cone photoreceptors. Loss of function of Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1, resulting in a radial displacement of horizontal cells that partially phenocopies the loss of function of Lhx1. These findings not only demonstrate that Spalt family transcription factors play a conserved role in regulating photoreceptor development in insects and mammals, but also identify Sall3 as a factor that regulates terminal differentiation of both cone photoreceptors and their postsynaptic partners.
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Affiliation(s)
- Jimmy de Melo
- Department of Neuroscience, Johns Hopkins University School of Medicine, 733 N. Broadway Avenue, Baltimore, MD 21287, USA
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41
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Johnston RJ, Otake Y, Sood P, Vogt N, Behnia R, Vasiliauskas D, McDonald E, Xie B, Koenig S, Wolf R, Cook T, Gebelein B, Kussell E, Nagakoshi H, Desplan C. Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye. Cell 2011; 145:956-68. [PMID: 21663797 PMCID: PMC3117217 DOI: 10.1016/j.cell.2011.05.003] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2010] [Revised: 03/25/2011] [Accepted: 05/05/2011] [Indexed: 12/31/2022]
Abstract
How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification.
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Affiliation(s)
- Robert J. Johnston
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
| | - Yoshiaki Otake
- Graduate School of Natural Science and Technology, Okayama University 3-1-1, Tsushima-naka, Okayama, Okayama 700-8530, Japan
| | - Pranidhi Sood
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
| | - Nina Vogt
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
| | - Rudy Behnia
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
| | - Daniel Vasiliauskas
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
| | - Elizabeth McDonald
- Division of Developmental Biology, Cincinnati Children’s Hospital, 3333 Burnet Ave, MLC 7007, Cincinnati, OH 45229
| | - Baotong Xie
- Division of Developmental Biology, Cincinnati Children’s Hospital, 3333 Burnet Ave, MLC 7007, Cincinnati, OH 45229
| | - Sebastian Koenig
- Rudolf-Virchow-Zentrum, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany
| | - Reinhard Wolf
- Rudolf-Virchow-Zentrum, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany
| | - Tiffany Cook
- Division of Developmental Biology, Cincinnati Children’s Hospital, 3333 Burnet Ave, MLC 7007, Cincinnati, OH 45229
| | - Brian Gebelein
- Division of Developmental Biology, Cincinnati Children’s Hospital, 3333 Burnet Ave, MLC 7007, Cincinnati, OH 45229
| | - Edo Kussell
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
| | - Hideki Nagakoshi
- Graduate School of Natural Science and Technology, Okayama University 3-1-1, Tsushima-naka, Okayama, Okayama 700-8530, Japan
| | - Claude Desplan
- Department of Biology, New York University, 100 Washington Square East, New York NY 10003, USA
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42
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Gambis A, Dourlen P, Steller H, Mollereau B. Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila. Dev Biol 2011; 351:128-34. [PMID: 21215264 PMCID: PMC3051417 DOI: 10.1016/j.ydbio.2010.12.040] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2010] [Revised: 12/22/2010] [Accepted: 12/22/2010] [Indexed: 11/25/2022]
Abstract
We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The "Tomato/GFP-FLP/FRT" method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.
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Affiliation(s)
- Alexis Gambis
- Howard Hughes Medical Institute, Laboratory of Apoptosis and Cancer, The Rockefeller University, New York, NY, USA
| | - Pierre Dourlen
- Laboratory of Molecular Biology of the Cell, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France
| | - Hermann Steller
- Howard Hughes Medical Institute, Laboratory of Apoptosis and Cancer, The Rockefeller University, New York, NY, USA
| | - Bertrand Mollereau
- Laboratory of Molecular Biology of the Cell, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France
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43
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Cook T, Zelhof A, Mishra M, Nie J. 800 facets of retinal degeneration. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2011; 100:331-68. [PMID: 21377630 DOI: 10.1016/b978-0-12-384878-9.00008-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
In today's world of genomics and large computational analyses, rapid progress has been made in identifying genes associated with human retinal diseases. Nevertheless, before significant advances toward effective therapeutic intervention is made, a clearer understanding of the molecular and cellular role of these gene products in normal and diseased photoreceptor cell biology is required. Given the complexity of the vertebrate retina, these advancements are unlikely to be revealed in isolated human cell lines, but instead, will require the use of numerous model systems. Here, we describe several parallels between vertebrate and invertebrate photoreceptor cell biology that are beginning to emerge and advocate the use of Drosophila melanogaster as a powerful genetic model system for uncovering molecular mechanisms of human retinal pathologies, in particular photoreceptor neurodegeneration.
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Affiliation(s)
- T Cook
- Department of Pediatric Ophthalmology, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA
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44
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Astigarraga S, Hofmeyer K, Treisman JE. Missed connections: photoreceptor axon seeks target neuron for synaptogenesis. Curr Opin Genet Dev 2010; 20:400-7. [PMID: 20434326 DOI: 10.1016/j.gde.2010.04.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2010] [Revised: 03/31/2010] [Accepted: 04/02/2010] [Indexed: 02/06/2023]
Abstract
Extending axons must choose the appropriate synaptic target cells in order to assemble functional neural circuitry. The axons of the Drosophila color-sensitive photoreceptors R7 and R8 project as a single fascicle from each ommatidium, but their terminals are segregated into distinct layers within their target region. Recent studies have begun to reveal the molecular mechanisms that establish this projection pattern. Both homophilic adhesion molecules and specific ligand-receptor interactions make important contributions to stabilizing R7 and R8 terminals in the appropriate target layers. These cell recognition molecules are regulated by the same transcription factors that control R7 and R8 cell fates. Autocrine and repulsive signaling mechanisms prevent photoreceptor terminals from encroaching on their neighbors, preserving the spatial resolution of visual information.
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Affiliation(s)
- Sergio Astigarraga
- Kimmel Center for Biology and Medicine of the Skirball Institute, Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA
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45
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Charlton-Perkins M, Cook TA. Building a fly eye: terminal differentiation events of the retina, corneal lens, and pigmented epithelia. Curr Top Dev Biol 2010; 93:129-73. [PMID: 20959165 DOI: 10.1016/b978-0-12-385044-7.00005-9] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
In the past, vast differences in ocular structure, development, and physiology throughout the animal kingdom led to the widely accepted notion that eyes are polyphyletic, that is, they have independently arisen multiple times during evolution. Despite the dissimilarity between vertebrate and invertebrate eyes, it is becoming increasingly evident that the development of the eye in both groups shares more similarity at the genetic level than was previously assumed, forcing a reexamination of eye evolution. Understanding the molecular underpinnings of cell type specification during Drosophila eye development has been a focus of research for many labs over the past 25 years, and many of these findings are nicely reviewed in Chapters 1 and 4. A somewhat less explored area of research, however, considers how these cells, once specified, develop into functional ocular structures. This review aims to summarize the current knowledge related to the terminal differentiation events of the retina, corneal lens, and pigmented epithelia in the fly eye. In addition, we discuss emerging evidence that the different functional components of the fly eye share developmental pathways and functions with the vertebrate eye.
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Affiliation(s)
- Mark Charlton-Perkins
- Department of Pediatric Ophthalmology, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA
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46
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Amin N, Khan A, St. Johnston D, Tomlinson I, Martin S, Brenman J, McNeill H. LKB1 regulates polarity remodeling and adherens junction formation in the Drosophila eye. Proc Natl Acad Sci U S A 2009; 106:8941-6. [PMID: 19443685 PMCID: PMC2690039 DOI: 10.1073/pnas.0812469106] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2008] [Indexed: 12/31/2022] Open
Abstract
The serine-threonine kinase LKB1 regulates cell polarity from Caenorhabditis elegans to man. Loss of lkb1 leads to a cancer predisposition, known as Peutz-Jeghers Syndrome. Biochemical analysis indicates that LKB1 can phosphorylate and activate a family of AMPK- like kinases, however, the precise contribution of these kinases to the establishment and maintenance of cell polarity is still unclear. Recent studies propose that LKB1 acts primarily through the AMP kinase to establish and/or maintain cell polarity. To determine whether this simple model of how LKB1 regulates cell polarity has relevance to complex tissues, we examined lkb1 mutants in the Drosophila eye. We show that adherens junctions expand and apical, junctional, and basolateral domains mix in lkb1 mutants. Surprisingly, we find LKB1 does not act primarily through AMPK to regulate cell polarity in the retina. Unlike lkb1 mutants, ampk retinas do not show elongated rhabdomeres or expansion of apical and junctional markers into the basolateral domain. In addition, nutrient deprivation does not reveal a more dramatic polarity phenotype in lkb1 photoreceptors. These data suggest that AMPK is not the primary target of LKB1 during eye development. Instead, we find that a number of other AMPK-like kinase, such as SIK, NUAK, Par-1, KP78a, and KP78b show phenotypes similar to weak lkb1 loss of function in the eye. These data suggest that in complex tissues, LKB1 acts on an array of targets to regulate cell polarity.
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Affiliation(s)
- Nancy Amin
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada M5G 1X5
| | - Afifa Khan
- Molecular and Population Genetics Laboratory, Cancer Research UK, London WC2A 3PX, United Kingdom; and
| | - Daniel St. Johnston
- Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom
| | - Ian Tomlinson
- Molecular and Population Genetics Laboratory, Cancer Research UK, London WC2A 3PX, United Kingdom; and
| | - Sophie Martin
- Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom
| | - Jay Brenman
- Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599
| | - Helen McNeill
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada M5G 1X5
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8
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47
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Birkholz DA, Chou WH, Phistry MM, Britt SG. rhomboid mediates specification of blue- and green-sensitive R8 photoreceptor cells in Drosophila. J Neurosci 2009; 29:2666-75. [PMID: 19261861 PMCID: PMC2679528 DOI: 10.1523/jneurosci.5988-08.2009] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2008] [Accepted: 01/16/2009] [Indexed: 01/09/2023] Open
Abstract
Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila there is growing evidence that the color sensitivity of the R8 cell within an individual ommatidium is regulated by an inductive signal from the adjacent R7 cell. We previously examined the retinal patterning defect in Scutoid mutants, which results from a disruption of rhomboid expression. Here we show that loss of rhomboid blocks the induction of Rh5 expression and misexpression of rhomboid leads to the inappropriate induction of Rh5. These effects are specific to rhomboid, because its paralogue roughoid is neither required nor sufficient for the induction of Rh5 expression. We show that rhomboid is required cell-autonomously within the R8 photoreceptor cells and nonautonomously elsewhere in the eye for Rh5 induction. Interestingly, we found that the Epidermal growth factor receptor is also required for Rh5 induction, and its activation is sufficient to rescue the loss of Rh5 induction in a rhomboid mutant. This suggests that rhomboid may function in R8 cells to activate Epidermal growth factor receptor signaling in R7 cells and promote their differentiation to a signaling competent state.
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Affiliation(s)
| | - Wen-Hai Chou
- Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, Texas 78245
| | | | - Steven G. Britt
- Department of Cell and Developmental Biology and
- Department of Ophthalmology and Rocky Mountain Lions Eye Institute, University of Colorado Denver, School of Medicine, Aurora, Colorado 80045, and
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48
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Bao R, Friedrich M. Molecular Evolution of the Drosophila Retinome: Exceptional Gene Gain in the Higher Diptera. Mol Biol Evol 2009; 26:1273-87. [DOI: 10.1093/molbev/msp039] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
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49
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SALL3 interacts with DNMT3A and shows the ability to inhibit CpG island methylation in hepatocellular carcinoma. Mol Cell Biol 2009; 29:1944-58. [PMID: 19139273 DOI: 10.1128/mcb.00840-08] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The mechanisms of aberrant CpG island methylation in oncogenesis are not fully characterized. In particular, little is known about the mechanisms of inhibition of CpG island methylation. Here we show that sal-like 3 (SALL3) is a novel inhibitory factor for DNA methyltransferase 3 alpha (DNMT3A). SALL3 binds to DNMT3A by a direct interaction between the double zinc finger motif of SALL3 and the PWWP domain of DNMT3A. SALL3 expression reduces DNMT3A-mediated CpG island methylation in cell culture and in vitro. CpG island methylation is enhanced in SALL3-depleted cells. Consistently, DNMT3A from SALL3-depleted cells increases methyltransferase activity in vitro. Binding of DNMT3A to chromatin is reduced or increased by SALL3 expression or depletion, respectively, accounting for the mechanism by which SALL3 inhibits DNMT3A-mediated CpG island methylation. We also show that SALL3 is inducible by BMP-4 and silenced by associated DNA methylation in hepatocellular carcinoma (HCC). Our results suggest that silencing of SALL3 results in acceleration of DNA methylation in HCC. This functional characterization of SALL3 sheds light on regulatory mechanisms for DNMT3A and provides new strategies to inhibit aberrant methylation in cancer.
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50
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Kiefer SM, Robbins L, Barina A, Zhang Z, Rauchman M. SALL1 truncated protein expression in Townes-Brocks syndrome leads to ectopic expression of downstream genes. Hum Mutat 2008; 29:1133-40. [PMID: 18470945 DOI: 10.1002/humu.20759] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Mutations in SALL1 lead to the dominant multiorgan congenital anomalies that define Townes-Brocks syndrome (TBS). The majority of these mutations result in premature termination codons that would be predicted to trigger nonsense-mediated decay (NMD) of mutant mRNA and cause haploinsufficiency. Our previous studies using a gene targeted mouse model (Sall1-DeltaZn) suggested that TBS phenotypes are due to expression of a truncated mutant protein, not haploinsufficiency. In this report, we strengthen this hypothesis by showing that expression of the mutant protein alone in transgenic mice is sufficient to cause limb phenotypes that are characteristic of TBS patients. We prove that the same pathogenetic mechanism elucidated in mice is occurring in humans by demonstrating that truncated SALL1 protein is expressed in cells derived from a TBS patient. TBS mutant protein is capable of dominant negative activity that results in ectopic activation of two downstream genes, Nppa and Shox2, in the developing heart and limb. We propose a model for the pathogenesis of TBS in which truncated Sall1 protein causes derepression of Sall-responsive target genes.
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Affiliation(s)
- Susan M Kiefer
- Research and Education Service Line, St. Louis Veterans Administration Medical Center, U.S. Department of Veterans Affairs, St. Louis, Missouri 63106, USA
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