Publications by authors named "Francesca Pignoni"

24 Publications

  • Page 1 of 1

STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the eye disc peripodial epithelium.

J Cell Sci 2020 05 26;133(10). Epub 2020 May 26.

Department of Ophthalmology and Visual Sciences, Upstate Medical University, 505 Irving Avenue, NRB 4610, Syracuse, NY 13210, USA

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified protein phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hippo kinase, and consequently its substrate Warts, to release the transcriptional co-activator Yorkie into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.
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http://dx.doi.org/10.1242/jcs.237834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272332PMC
May 2020

Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components.

BMC Dev Biol 2019 01 22;19(1). Epub 2019 Jan 22.

Center for Vision Research and Department of Ophthalmology, Upstate Medical University, NRB-4610, 505 Irving Ave, Syracuse, 13210, NY, USA.

Background: BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to our understanding of its molecular and genetic mechanisms. These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments.

Results: Herein we identify and characterize the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, we report complex regulatory feedback on core pathway genes in this system.

Conclusions: Native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. We propose that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.
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http://dx.doi.org/10.1186/s12861-019-0181-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341649PMC
January 2019

Mutant analysis by rescue gene excision: New tools for mosaic studies in Drosophila.

Genesis 2016 Nov 21;54(11):589-592. Epub 2016 Oct 21.

Departments of Ophthalmology, Center for Vision Research and SUNY Eye Institute SUNY Upstate Medical University, Syracuse, New York, USA.

A host of classical and molecular genetic tools make Drosophila a tremendous model for the dissection of gene activity. In particular, the FLP-FRT technique for mitotic recombination has greatly enhanced gene loss-of-function analysis. This technique efficiently induces formation of homozygous mutant clones in tissues of heterozygous organisms. However, the dependence of the FLP-FRT method on cell division, and other constraints, also impose limits on its effectiveness. We describe here the generation and testing of tools for Mutant Analysis by Rescue Gene Excision (MARGE), an approach whereby mutant cells are formed by loss of a rescue transgene in a homozygous mutant organism. Rescue-transgene loss can be induced in any tissue or cell-type and at any time during development or in the adult using available heat-shock-induced or tissue-specific flippases, or combinations of UAS-FLP with Gal4 and Gal80 reagents. The simultaneous loss of a constitutive fluorescence marker (GFP or RFP) identifies the mutant cells. We demonstrate the efficacy of the MARGE technique by flip-out (clonal and disc-wide) of a Ubi-GFP-carrying construct in imaginal discs, and by inducing a known yki mutant phenotype in the Drosophila ovary.
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http://dx.doi.org/10.1002/dvg.22984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357640PMC
November 2016

Distinct regulation of atonal in a visual organ of Drosophila: Organ-specific enhancer and lack of autoregulation in the larval eye.

Dev Biol 2017 Jan 29;421(1):67-76. Epub 2016 Sep 29.

Department of Ophthalmology and Center for Vision Research, SUNY Upstate Medical University, Syracuse, NY, USA; Departments of Neuroscience & Physiology and Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA. Electronic address:

Drosophila has three types of visual organs, the larval eyes or Bolwig's organs (BO), the ocelli (OC) and the compound eyes (CE). In all, the bHLH protein Atonal (Ato) functions as the proneural factor for photoreceptors and effects the transition from progenitor cells to differentiating neurons. In this work, we investigate the regulation of ato expression in the BO primordium (BOP). Surprisingly, we find that ato transcription in the BOP is entirely independent of the shared regulatory DNA for the developing CE and OC. The core enhancer for BOP expression, ato, lies ~6kb upstream of the ato gene, in contrast to the downstream location of CE and OC regulatory elements. Moreover, maintenance of ato expression in the neuronal precursors through autoregulation-a common and ancient feature of ato expression that is well-documented in eyes, ocelli and chordotonal organs-does not occur in the BO. We also show that the ato enhancer contains two binding sites for the transcription factor Sine oculis (So), a core component of the progenitor specification network in all three visual organs. These binding sites function in vivo and are specifically bound by So in vitro. Taken together, our findings reveal that the control of ato transcription in the evolutionarily derived BO has diverged considerably from ato regulation in the more ancestral compound eyes and ocelli, to the extent of acquiring what appears to be a distinct and evolutionarily novel cis-regulatory module.
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http://dx.doi.org/10.1016/j.ydbio.2016.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358805PMC
January 2017

Shared and distinct mechanisms of atonal regulation in Drosophila ocelli and compound eyes.

Dev Biol 2016 10 23;418(1):10-16. Epub 2016 Aug 23.

Department of Ophthalmology and Center for Vision Research, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Department of Neuroscience & Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA. Electronic address:

The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, our findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye.
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http://dx.doi.org/10.1016/j.ydbio.2016.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223745PMC
October 2016

Fly LMBR1/LIMR-type protein Lilipod promotes germ-line stem cell self-renewal by enhancing BMP signaling.

Proc Natl Acad Sci U S A 2015 Nov 28;112(45):13928-33. Epub 2015 Oct 28.

Department of Biochemistry and Molecular Biology, Upstate Medical University, Syracuse, NY 13210; Department of Ophthalmology and Center for Vision Research, Upstate Medical University, Syracuse, NY 13210; Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, NY 13210

Limb development membrane protein-1 (LMBR1)/lipocalin-interacting membrane receptor (LIMR)-type proteins are putative nine-transmembrane receptors that are evolutionarily conserved across metazoans. However, their biological function is unknown. Here, we show that the fly family member Lilipod (Lili) is required for germ-line stem cell (GSC) self-renewal in the Drosophila ovary where it enhances bone morphogenetic protein (BMP) signaling. lili mutant GSCs are lost through differentiation, and display reduced levels of the Dpp transducer pMad and precocious activation of the master differentiation factor bam. Conversely, overexpressed Lili induces supernumerary pMad-positive bamP-GFP-negative GSCs. Interestingly, differentiation of lili mutant GSCs is bam-dependent; however, its effect on pMad is not. Thus, although it promotes stem cell self-renewal by repressing a bam-dependent process, Lilipod enhances transduction of the Dpp signal independently of its suppression of differentiation. In addition, because Lili is still required by a ligand-independent BMP receptor, its function likely occurs between receptor activation and pMad phosphorylation within the signaling cascade. This first, to our knowledge, in vivo characterization of a LMBR1/LIMR-type protein in a genetic model reveals an important role in modulating BMP signaling during the asymmetric division of an adult stem cell population and in other BMP signaling contexts.
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http://dx.doi.org/10.1073/pnas.1509856112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653181PMC
November 2015

Using Xenopus to discover new genes involved in branchiootorenal spectrum disorders.

Comp Biochem Physiol C Toxicol Pharmacol 2015 Dec 24;178:16-24. Epub 2015 Jun 24.

Department of Ophthalmology, Upstate Medical University, Syracuse, NY, USA.

Congenital hearing loss is an important clinical problem because, without early intervention, affected children do not properly acquire language and consequently have difficulties developing social skills. Although most newborns in the US are screened for hearing deficits, even earlier diagnosis can be made with prenatal genetic screening. Genetic screening that identifies the relevant mutated gene can also warn about potential congenital defects in organs not related to hearing. We will discuss efforts to identify new candidate genes that underlie the Branchiootorenal spectrum disorders in which affected children have hearing deficits and are also at risk for kidney defects. Mutations in two genes, SIX1 and EYA1, have been identified in about half of the patients tested. To uncover new candidate genes, we have used the aquatic animal model, Xenopus laevis, to identify genes that are part of the developmental genetic pathway of Six1 during otic and kidney development. We have already identified a large number of potential Six1 transcriptional targets and candidate co-factor proteins that are expressed at the right time and in the correct tissues to interact with Six1 during development. We discuss the advantages of using this system for gene discovery in a human congenital hearing loss syndrome.
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http://dx.doi.org/10.1016/j.cbpc.2015.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662879PMC
December 2015

Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1.

J Cell Sci 2015 Aug 19;128(15):2938-50. Epub 2015 Jun 19.

Department of Ophthalmology, Center for Vision Research and SUNY Eye Institute, Upstate Medical University, Syracuse, 13210 NY, USA Departments of Neuroscience and Physiology, Biochemistry and Molecular Biology, Upstate Medical University, Syracuse, 13210 NY, USA

The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis.
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http://dx.doi.org/10.1242/jcs.173807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540953PMC
August 2015

ato-Gal4 fly lines for gene function analysis: Eya is required in late progenitors for eye morphogenesis.

Genesis 2015 Jun 10;53(6):347-55. Epub 2015 Jun 10.

Department of Ophthalmology, Center for Vision Research, and SUNY Eye Institute, SUNY Upstate Medical University, Syracuse, New York.

The Gal4/UAS system is one of the most powerful tools for the study of cellular and developmental processes in Drosophila. Gal4 drivers can be used to induce targeted expression of dominant-negative and dominant-active proteins, histological markers, activity sensors, gene-specific dsRNAs, modulators of cell survival or proliferation, and other reagents. Here, we describe novel atonal-Gal4 lines that contain regions of the regulatory DNA of atonal, the proneural gene for photoreceptors, stretch receptors, auditory organ, and some olfactory sensilla. During neurogenesis, the atonal gene is expressed at a critical juncture, a time of transition from progenitor cell to developing neuron. Thus, these lines are particularly well suited for the study of the transcription factors and signaling molecules orchestrating this critical transition. To demonstrate their usefulness, we focus on two visual organs, the eye and the Bolwig. We demonstrate the induction of predicted eye phenotypes when expressing the dominant-negative EGF receptor or a dsRNA against Notch in the developing eye disc. In another example, we show the deletion of the Bolwig's organ using the proapoptotic factor Hid. Finally, we investigate the function of the eye specification factor Eyes absent or Eya in late retinal progenitors, shortly before they begin morphogenesis. We show that Eya is still required in these late progenitors to promote eye formation, and show failure to induce the target gene atonal and consequent lack of neuron formation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063080PMC
http://dx.doi.org/10.1002/dvg.22858DOI Listing
June 2015

Onset of atonal expression in Drosophila retinal progenitors involves redundant and synergistic contributions of Ey/Pax6 and So binding sites within two distant enhancers.

Dev Biol 2014 Feb 16;386(1):152-64. Epub 2013 Nov 16.

Department of Ophthalmology, Center for Vision Research, and SUNY Eye Institute, SUNY Upstate Medical University, Syracuse, NY, USA; Departments of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA; Department of Neuroscience & Physiology, SUNY Upstate Medical University, Syracuse, NY, USA. Electronic address:

Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. We show here that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). We show how these sites interact to induce ato expression in distinct regions of the eye field and confirm them to be occupied by endogenous Ey and So proteins in vivo. Our study suggests that Ey and So operate differently through the same 3' cis-regulatory sites in distinct populations of retinal progenitors.
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http://dx.doi.org/10.1016/j.ydbio.2013.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223736PMC
February 2014

Can fly photoreceptors lead to treatments for rho ((P23H)) -linked retinitis pigmentosa?

J Ophthalmic Vis Res 2013 Jan;8(1):86-91

SUNY Eye Institute, Department of Ophthalmology and Department of Neuroscience and Physiology, and Center for Vision Research SUNY Upstate Medical University, Syracuse, NY, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691985PMC
January 2013

Homeostasis of the Drosophila adult retina by actin-capping protein and the Hippo pathway.

Commun Integr Biol 2011 Sep 1;4(5):612-5. Epub 2011 Sep 1.

Instituto Gulbenkian de Ciência; Oeiras, Portugal.

The conserved Hippo signaling pathway regulates multiple cellular events, including tissue growth, cell fate decision and neuronal homeostasis. While the core Hippo kinase module appears to mediate all the effects of the pathway, various upstream inputs have been identified depending on tissue context. We have recently shown that, in the Drosophila wing imaginal disc, actin-Capping Protein and Hippo pathway activities inhibit F-actin accumulation. In turn, the reduction in F-actin sustains Hippo pathway activity, preventing Yorkie nuclear translocation and the upregulation of proliferation and survival genes. Here, we investigate the role of Capping Protein in growth-unrelated events controlled by the Hippo pathway. We provide evidence that loss of Capping Protein induces degeneration of the adult Drosophila retina through misregulation of the Hippo pathway. We propose a model by which F-actin dynamics might be involved in all processes that require the activity of the core Hippo kinase module.
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http://dx.doi.org/10.4161/cib.4.5.16853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204144PMC
September 2011

Identification of Bombyx atonal and functional comparison with the Drosophila atonal proneural factor in the developing fly eye.

Genesis 2012 May 27;50(5):393-403. Epub 2011 Dec 27.

Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Science, Zhejiang University, Hangzhou 310029, China.

The proneural genes are fundamental regulators of neuronal development in all metazoans. A critical role of the fly proneural factor Atonal (Ato(Dm)) is to induce photoreceptor neuron formation in Drosophila, whereas its murine homolog, Atonal7(Mm) (aka Ath5) is essential for the development of the ganglion cells of the vertebrate eye. Here, we identify the Bombyx mori ato homolog (ato(Bm) ). In a pattern strikingly reminiscent of ato(Dm), the ato(Bm) mRNA is expressed as a stripe in the silkworm eye disc. Its DNA-binding and protein-protein interaction domain is highly homologous to the Ato(Dm) bHLH. Targeted expression of Ato(Bm) in the endogenous ato(Dm) pattern rescues the eyeless phenotype of the fly ato(1) mutant and its ectopic expression induces similar gain-of-function phenotypes as Ato(Dm). Rescue experiments with chimeric proteins show that the non-bHLH portion of Ato(Bm) (N-region) can effectively substitute for the corresponding region of the fly transcription factor, even though no apparent conservation can be found at the amino acid level. On the contrary, the highly similar bHLH domain of Ato(Bm) cannot similarly substitute for the corresponding region of Ato(Dm). Thus, the bHLH(Bm) domain requires the Ato(Bm) N-region to function effectively, whereas the bHLH(Dm) domain can operate well with either N-region. These findings suggest a role for the non-bHLH portion of Ato proteins in modulating the function of the bHLH domain in eye neurogenesis and implicate specific aa residues of the bHLH in this process.
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http://dx.doi.org/10.1002/dvg.20816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362925PMC
May 2012

Tubby-RFP balancers for developmental analysis: FM7c 2xTb-RFP, CyO 2xTb-RFP, and TM3 2xTb-RFP.

Genesis 2012 Feb 6;50(2):119-23. Epub 2012 Jan 6.

Department of Ophthalmology, Center for Vision Research, SUNY Eye Institute, SUNY Upstate Medical University, Syracuse, New York 13210, USA.

We report here the construction of Tubby-RFP balancers for the X, 2nd and 3rd chromosomes of Drosophila melanogaster. The insertion of a 2xTb-RFP transgene on the FM7c, CyO, and TM3 balancer chromosomes introduces two easily scorable, dominant, developmental markers. The strong Tb phenotype is visible to the naked eye at the larval L2, L3, and pupal stages. The RFP associated with the cuticle is easily detected at all stages from late embryo to adult with the use of a fluorescence stereomicroscope. The FM7c Bar 2xTb-RFP, CyO Cy 2xTb-RFP, and TM3 Sb 2xTb-RFP balancers will greatly facilitate the analysis of lethals and other developmental mutants in L2/L3 larvae and pupae, but also provide coverage of other stages beginning in late embryogenesis through to the adult.
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http://dx.doi.org/10.1002/dvg.20801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931234PMC
February 2012

Yki/YAP, Sd/TEAD and Hth/MEIS control tissue specification in the Drosophila eye disc epithelium.

PLoS One 2011 19;6(7):e22278. Epub 2011 Jul 19.

Department of Ophthalmology, Center for Vision Research, and SUNY Eye Institute, SUNY Upstate Medical University, Syracuse, New York, United States of America.

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. We show here that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc epithelium at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. We also show that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. Our results support a critical role for Yki- and its partners Sd and Hth--in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022278PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139632PMC
December 2011

Developmental expression patterns of candidate cofactors for vertebrate six family transcription factors.

Dev Dyn 2010 Dec;239(12):3446-66

Department of Anatomy and Regenerative Biology, The George Washington University, School of Medicine and Health Sciences, Washington, DC 20037, USA.

Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by cofactor proteins. Two Six genes and one cofactor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for approximately half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila cofactor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes.
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http://dx.doi.org/10.1002/dvdy.22484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059517PMC
December 2010

Analysis of the Otd-dependent transcriptome supports the evolutionary conservation of CRX/OTX/OTD functions in flies and vertebrates.

Dev Biol 2008 Mar 31;315(2):521-34. Epub 2008 Jan 31.

Department of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, 243 Charles Street MEEI 507, Boston, MA 02445, USA.

Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins, CRX, OTX1, and OTX2, fulfill these functions. In Drosophila, the single founding member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. We have used global gene expression analysis in late pupal heads to better characterize the post-embryonic functions of Otd in Drosophila. We have identified 61 genes that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one-third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well-documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes, notwithstanding the significant divergence of the underlying genetic pathways. Our findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria.
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http://dx.doi.org/10.1016/j.ydbio.2007.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2329912PMC
March 2008

Direct control of neurogenesis by selector factors in the fly eye: regulation of atonal by Ey and So.

Development 2006 Dec 15;133(24):4881-9. Epub 2006 Nov 15.

Ophthalmology Department, Harvard Medical School/MEEI, Boston, MA 02114, USA.

During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of ;eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. We show here that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. We find that these transcription factors interact in vitro and provide indirect evidence that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation.
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http://dx.doi.org/10.1242/dev.02669DOI Listing
December 2006

Nicastrin controls aspects of photoreceptor neuron specification and differentiation in the Drosophila eye.

Dev Dyn 2005 Nov;234(3):590-601

Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.

Nicastrin is a component of the Notch signaling pathway involved in proteolytic release of the Notch receptor intracellular domain. It has been postulated that intracellular Notch is required within the nucleus of fly eye progenitor cells to enhance (pro-neural enhancement) and then repress (lateral inhibition) transcription of pro-neural genes. We present here an analysis of Nicastrin function during eye development and find that Nicastrin is essential to early photoreceptor neuron development. Nicastrin mutant tissue displays neuronal loss or hyperplasia; these phenotypes can be rescued by targeted expression of an intracellular form of Notch. Thus, nuclear translocation of Notch and its direct regulation of gene expression appear to be critical to pro-neural enhancement as well as lateral inhibition. In addition, we show that Nicastrin as well as Notch are required to maintain normal R-cell morphology, because the nuclei of mutant photoreceptor neurons cannot maintain their proper position. Thus, Notch signaling plays a role, not only in cell fate specification, but also in differentiation of photoreceptor neurons.
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http://dx.doi.org/10.1002/dvdy.20543DOI Listing
November 2005

Partner specificity is essential for proper function of the SIX-type homeodomain proteins Sine oculis and Optix during fly eye development.

Dev Biol 2005 Oct;286(1):158-68

Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA.

The development of the Drosophila visual system utilizes two members of the highly conserved Six-Homeobox family of transcription factor, Sine oculis and Optix. Although in vitro studies have detected differences in DNA-binding and interactions with some co-factors, questions remain as to what extent the activity for these two transcriptional regulators is redundant or specific in vivo. In this work, we show that the SoD mutation within the Six domain does not abolish DNA-protein interactions, but alters co-factor binding specificity to resemble that of Optix. A mutation in the same region of Optix alters its activity in vivo. We propose that the dominant mutant phenotype is primarily due to an alteration in binding properties of the Sine oculis protein and that distinct partner interactions is one important mechanism in determining significant functional differences between these highly conserved factors during eye development.
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http://dx.doi.org/10.1016/j.ydbio.2005.07.017DOI Listing
October 2005

Fly SIX-type homeodomain proteins Sine oculis and Optix partner with different cofactors during eye development.

Dev Dyn 2005 Nov;234(3):497-504

Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.

Two members from the Six class of homeobox transcription factors, Sine oculis (SO) and Optix, function during development of the fly visual system. Differences in gain-of-function phenotypes and gene expression suggest that these related factors play distinct roles in the formation of the fly eye. However, the molecular nature of their functional differences remains unclear. In this study, we report the identification of two novel factors that participate in specific partnerships with Sine oculis and Optix during photoreceptor neurons formation and in eye progenitor cells. This work shows that different cofactors likely mediate unique functions of Sine oculis and Optix during the development of the fly eye and that the repeated requirement for SO function at multiple stages of eye development reflects the activity of different SO-cofactor complexes.
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http://dx.doi.org/10.1002/dvdy.20442DOI Listing
November 2005

Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor.

Development 2004 Dec 3;131(23):5871-81. Epub 2004 Nov 3.

Department of Anatomy and Cell Biology, Institute for Biomedical Sciences, The George Washington University, Washington, DC 20037, USA.

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. We have used markers of pre-placodal ectoderm (PPE) (six1, eya1) to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.
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http://dx.doi.org/10.1242/dev.01516DOI Listing
December 2004

The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development.

Genetics 2004 May;167(1):233-41

Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavík, Iceland.

The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1470875PMC
http://dx.doi.org/10.1534/genetics.167.1.233DOI Listing
May 2004

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head.

Dev Cell 2003 Sep;5(3):403-14

Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA, 02114, USA.

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). Here, we investigate the function of these signaling pathways in this process. We find that organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Our analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. We propose that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development.
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http://dx.doi.org/10.1016/s1534-5807(03)00243-0DOI Listing
September 2003