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Pax6 A multi level regulator of ocular developmentq Ohad Shaham a Yotam Menuchina Chen Farhya Ruth Ashery Padana b aSackler Faculty of Medicine Department of Human Molecular Genetics and Biochemistry Tel Aviv University Ramat Aviv Tel Aviv 69978 Israel bSagol School of Neuroscience Tel Aviv University Ramat Aviv Tel Aviv 69978 Israel a r t i c l e i n f o Article history Available online 3 May 2012 Keywords Pax6 Lens Retina Eye development a b s t r a c t Eye development has been a paradigm for the study of organogenesis from the demonstration of lens induction through epithelial tissue morphogenesis to neuronal specifi cation and differentiation The transcription factor Pax6 has been shown to play a key role in each of these processes Pax6 is required for initiation of developmental pathways patterning of epithelial tissues activation of tissue specifi c genes and interaction with other regulatory pathways Herein we examine the data accumulated over the last few decades from extensive analyses of biochemical modules and genetic manipulation of the Pax6 gene Specifi cally we describe the regulation of Pax6 s expression pattern the protein s DNA binding properties and its specifi c roles and mechanisms of action at all stages of lens and retinal development Pax6 functions at multiple levels to integrate extracellular information and execute cell intrinsic differentiation programs that culminate in the specifi cation and differentiation of a distinct ocular lineage 2012 Elsevier Ltd All rights reserved Contents 1 The Pax6 genedhistorical perspective 352 2 Regulation and products of the Pax6 gene 352 2 1 Regulation of the complex expression profile of Pax6 352 2 2 Structure and transcriptional activity of the Pax6 proteins 354 3 The roles of Pax6 in specifi cation and morphogenesis of the eye 357 3 1 Specification of OV progenitors 357 3 1 1 Pax6 is one of several eye fi eld genes 357 3 1 2 Roles for EFTFs in the partitioning of the single eye field and early patterning of the OV 358 3 1 3 Extrinsic cues pattern the OV 359 3 2 The multiple functions of Pax6 in the development of OC derivatives 360 3 2 1 The development of the OC neuroepithelium to the ocular lineages 360 3 2 2 Roles for Pax6 in the differentiation of OC progenitors 361 3 2 3 Pax6 in RPC cell cycle regulation 363 3 3 Specification of the ocular surface ectoderm 364 3 3 1 Pax6 determines the lens pre placodal region 364 3 3 2 Extrinsic cues in lens fate determination 364 Abbreviations bHLH basic helix loop helix BMPs bone morphogenetic proteins BrdU bromodeoxyuridine CB ciliary body CNS central nervous system E embryonic day days post conception ECM extracellular matrix EE ectodermal enhancer EFTF eye fi eld transcription factor FGF fi broblast growth factor HH Hamburger and Hamilton HTH helix turn helix LE lens ectoderm LFC lens fi ber cell LP lens placode MAPK mitogen activated protein kinase OC optic cup OV optic vesicle P post natal day PAX paired box PE pigmented epithelium PPR pre placodal region PST proline serine threonine RPC retinal progenitor cell RPE retinal pigmented epithelium SE surface ectoderm SELEX systematic evolution of ligands by exponential enrichment TGFb transforming growth factorb YAC yeast artifi cial chromosome q Percentage of work contributed by each author in the production of the manuscript is as follows Ohad Shaham contributed to sections 1 3 3 3 4 and Fig 5 Yotam Menuchin contributed section 2 and Fig 1 Chen Farhy contributed to section 3 2 and Figs 3 and 4 Ruth Ashery Padan incorporated the different parts wrote other sections and prepared Fig 2 Corresponding author Sackler Faculty of Medicine Department of Human Molecular Genetics and Biochemistry Tel Aviv University Ramat Aviv Tel Aviv 69978 Israel Tel 972 36409331 fax 972 36405834 E mail addresses ruthash post tau ac il asherypadan R Ashery Padan Contents lists available at SciVerse ScienceDirect Progress in Retinal and Eye Research journal homepage 1350 9462 e see front matter 2012 Elsevier Ltd All rights reserved doi 10 1016 j preteyeres 2012 04 002 Progress in Retinal and Eye Research 31 2012 351e376 The neural crest inhibits lens fate through negative regulation of Pax6 364 The role of the OV in lens induction 365 3 3 3 Pax6 is required in a stage dependent manner in the transition of PPR to lens placode 365 3 3 4 Morphogenesis of the lens placode and lens pit 366 3 3 5 A diploid level of Pax6 is required for detachment of the lens vesicle from the surface ectoderm 366 3 4 Roles of Pax6 in the transition from lens progenitors to differentiated cells 367 3 4 1 Lens fiber differentiation 367 3 4 2 Pax6 is required for differentiation of secondary lens fi bers 368 3 4 3 Pax6 down regulation is required at the fi nal stages of lens differentiation 368 3 5 Roles of Pax6 in corneal development and homeostasis 368 4 Conclusions and future directions 369 Acknowledgments 370 Supplementary data 370 References 370 1 The Pax6 genedhistorical perspective Long before the discovery of the Pax6 gene and its role in mammals spontaneously established Drosophila mutant lines in which eyes completely failed to develop were described and termed eyeless ey The eyeless phenotype served as a genetic marker and was mapped to chromosome 4 of Drosophila mela nogaster Sturtevant 1951 More than 10 years later and inde pendently of eyeless the semi dominant and homozygous lethal Small Eye Sey mutation was discovered in mice Roberts 1967 Observations of Sey Sey homozygous mouse embryos showed that the optic vesicle OV extends laterally during early gestation but the head surface ectoderm SE fails to develop into a lens aborting eye morphogenesis Hogan et al 1986 In heterozygous Sey mice the lens did develop but it was smaller and often remained connected to the cornea Hogan et al 1986 In humans a semi dominant condition termed congenital aniridia lack of iris was described long before modern genetic analysesbecame available Rush 1926 Aniridia is a recessive lethal disease which in the semi dominant heterozygous state involves a plethora of ocular abnormalities iris and ciliary body CB hypoplasia cataract and dislocation of the lens corneal opacity foveal dysplasia glaucoma and additional pathological phenotypes Lee et al 2008 Based on gene mapping evolutionary conservation and pheno typic similarities it was proposed that mouse Sey and human aniridia result from mutations in orthologous genes Glaser et al 1990 van der Meer de Jong et al 1990 The human aniridia gene wasisolatedbypositionalcloningandwasfoundtocontainapaired box Ton et al 1991 The paired box encodes a DNA binding domain that was initially identifi ed in Drosophila segmentation and subsequently discovered in a family of pairedbox containing mammalian genes given the name PAX Kessel and Gruss 1990 Treisman et al 1991 Walther et al 1991 As a member of the PAX family Pax6 was shown to be expressed in the murine central nervous system CNS eye olfactory system and pancreas Walther and Gruss 1991 It was subsequently demonstrated that the mouse Sey results from mutations in the Pax6 gene Hill et al 1991 In the following decades hundreds of causative mutations of aniridia were reported within the PAX6 locus The Human PAX6 mutation Databaseseehttp lsdb hgu mrc ac uk Brownetal 1998 Kokotas and Petersen 2010 Tzoulaki et al 2005 In parallel homologues of mammalian Pax6 were cloned in zebrafi sh and quail Krauss et al 1991 Martin et al 1992 Astound ingly conservation of Pax6 far exceeds the mammalian or even vertebratecontext UsingsequencehomologyofthemurinePax6gene Quiring et al 1994 were able to identify a paired and homeobox containinggenewithintheeyelesslocusofD melanogaster uncovering the causative mutations of the eyeless phenotype inside this gene s coding region Conservation of Pax6 is not limited to the DNA level Ectopic expressionof human Pax6in DrosophilaandXenopusembryos leads to the formation of displaceddyet apparently functional eye structures Chow et al 1999 Halder et al 1995 Viczian et al 2009 Zuber et al 2003 This indicates that there is a highly conserved genetic pathway triggered by Pax6 in combination with other eye transcription factors which is able to confer eye forming competence to certain embryonic tissues What remains a mystery is what aspects in Pax6 s activity are conserved among the different species Kozmik 2005 Another related question is how a seemingly conserved protein such as Pax6 regulates the diversity of cell lineages that populate the vertebrate eye Arendt et al 2009 Resolving these questions will contribute to our understanding of the molecular mechanisms regulating cell diversifi cation and organogenesis and of how the regulatory networks evolved from the most primitive meta zoaneyestoseveralremarkablydifferenteyestructures includingthe compound eyes of insects and the camera eyes of vertebrates 2 Regulation and products of the Pax6 gene 2 1 Regulation of the complex expression profi le of Pax6 In addition to its role in eye development Pax6 is pivotal for normal development of the CNS olfactory system and pancreas and plays a role in adult neurogenesis reviewed in Hanson and Van Heyningen 1995 Osumi et al 2008 Simpson and Price 2002 In each of these developmental contexts Pax6 displays a highly complex spatiotemporal expression patternwith a varied dosage of gene expression The dynamic and complex expression pattern of Pax6 which will be discussed in detail in the following sections can be attributed to regulatory DNA sequences that function in a coor dinated sometimes overlapping manner to tightly and robustly regulate gene expression The mammalian Pax6 gene encompasses 16 exons spanning roughly 28 kb 14 exons are numbered 0e13 and the other two are termedaand 5a Fig 1A black rectangles Glaser et al 1992 Kammandel et al 1999 Kim and Lauderdale 2006 Several different transcripts are synthesized at the Pax6 locus either due to selection of different promoters or through post transcriptional alternative splicing Two of the main transcription start sitesdP0 and P1 Plaza et al 1995b Xu and Saunders 1997 dencode two different 13 exon long transcripts that are translated into an identical polypeptide as translation of Pax6 begins at the fi rst ATG on exon 4 Walther and Gruss 1991 However P0 and P1 derived transcripts are differ entially regulated during embryonic development as evidenced by distinctspatialandtemporalexpressionpatternsofeach O Shaham et al Progress in Retinal and Eye Research 31 2012 351e376352 alternative transcript in the eye and brain Anderson et al 2002 Xu et al 1999b The biological signifi cance of P0 and P1 derived transcripts is not yet understood Nevertheless it might be that the existence of two separate promoters for the same protein allows for intricate regulation of the gene as each promoter may have its own set of adjacent regulatory sequences relieving the evolutionary constraints on each promoter A third promoter and transcription start site are located in an intronic sequence between exons 4 and 5 Termed promotera Pa Fig 1A this site initiates transcription of an alternative transcript which encodes a trun cated Pax6 protein variant Pax6DPD see section 2 2 Transcription from the above promoters is controlled by various enhancer sequences with remarkable tissue specifi city Utilizing sequence conservation DNaseI protection assays in vitro reporter transfections and transgenic reporter constructs several groups have identifi ed a multitude of regulatory sequences in the Pax6 locus Kammandel et al 1999 Kleinjan et al 2004 Plaza et al 1995a Williams et al 1998 Xu et al 1999b summarized in Fig 1A and Table 1 The cis elements located upstream of the Pax6 gene and in introns were initially identifi ed through a study of evolutionarily conserved regions that were functionally tested using transgenic Table 1 Tissue specifi c Pax6 enhancers ReferencesReporter expression patternApproximate pair coordinates relative to mouse P0 Code in Fig 1 Kammandel et al 1999Pancreatic islets 4 6ka Kammandel et al 1999 Williams et al 1998 Pax6 ectodermal enhancer EE surface ectoderm derivatives cornea lens conjunctiva lacrimal gland 3 9kb Xu et al 1999bPancreatic islets retinal progenitor cells 2 3kc Kammandel et al 1999dorsal telencephalon hindbrain spinal cord1 5e6 5kd Xu et al 1999bPhotoreceptor progenitors3 5ke Xu et al 1999b Plaza et al 1995a Kammandel et al 1999 Distal retinaa amacrine cells ciliary body iris14kf Kleinjan et al 2004Late eye development17 5kg Diencephalon19kh Rhombencephalon21ki Griffi n et al 2002Pretectum neural retina and olfactory regionw105k 106k and 107kJ k l Kleinjan et al 2006 2001Lens diencephalon hindbrain proximal retina cerebellumw110km Kleinjan et al 2001Telencephalon neuroretina retinal pigmented epitheliumw128kn Kleinjan et al 2006Forebrain diencephalon pineal glandw165ko Fig 1 Gene transcript and protein structure of Pax6 A Genomic structure of the mouse Pax6 gene not to scale The coding exons are colored the non coding exons are in black The regulatory elements are gray rhomboids and are designated with lowercase letters see Table 1 Transcription start sites are marked with arrows B The structure of Pax6 Pax6 5a proteins Phosphorylation sites are marked with an asterisk and the sumoylation site is marked with a number sign The HIPK2 and MAPK phosphorylation sites are colored blue and yellow respectively The amino acid positions correspond to the canonical Pax6 protein In A and B the paired domain and the exons that give rise to it are in red the glycine rich linker and the exons that give rise to it are in green the homeodomain and the exons that give rise to it are in purple and the PST domain and the exons that give rise to it are in light blue C Consensus SELEX driven binding sites of canonical Pax6 paired domain P6CON Pax6 5a paired domain 5aCON and Pax6 homeodomain P3 and site 2 1 which is bound by the homeodomain HD the PAI b sheets and linker regions Above the binding sequences are schematic representations of the Pax6 domains that bind them N any nucleotide W A or T S G or C K G or T M A or C Y T or C O Shaham et al Progress in Retinal and Eye Research 31 2012 351e376353 reporterassays Kammandel et al 1999 Williams et al 1998 Such analyses demonstrated distinct yet partly overlapping regulatory activity which simulated part of the total pattern of the intact gene For example an enhancer in intron 4 as well as cis elements upstream of P0 were found to activate gene expression in the developing retina regions f and c in Fig 1A Kammandel et al 1999 Xu et al 1999b and two distinct regions were identifi ed as activating reporter expression in the pancreas regions a and c in Fig 1A Xu et al 1999b Additional regulatory sequences were found near the proximal P1 promoter which are important for expression of the P1 transcript in the CNS and photoreceptors Table 1 and regions d and e in Fig 1A Kammandel et al 1999 Plaza et al 1995a Xu et al 1999b Thus Pax6 expression in a specifi c location is mediated by the combined activity of several transcriptional control elements This was exemplifi ed by the decrease but not elimination of Pax6 expression upon deletion of a lens enhancer Dimanlig et al 2001 region b in Fig 1A The overlapping activity of regulatory regions allows intricate regulation of expression levels in defi ned tissues and developmental time windows and also provides redundancy that contributes to robustness in gene activity An added benefi t of identifying Pax6 regulatory modules is their use for the establish ment of tissue specifi c Cre lines such as the transgenes Le Cre including regions a b c in Fig 1A Ashery Padan et al 2000 and a Cre region f Fig 1A Marquardt et al 2001 which are widely used for the study of gene function using Cre loxP technology in the lens and retina respectively In addition to the regulatory elements located upstream and within the introns more distant cis elements have been identifi ed downstream of the gene These came to light through compre hensive analyses of human aniridia patients who were carriers of deletions and chromosomal rearrangements Crolla and van Heyningen 2002 Fantes et al 1995 Lauderdale et al 2000 The regulatory elements were located downstream of the most distal breakpoint reported in human aniridia SIMO Lauderdale et al 2000 Simola et al 1983 These elements have been character ized in vivo using human yeast artifi cial chromosome YAC trans genic studies One such YAC encompassing 420 kb including the Pax6 encoding sequence was able torescuehomozygous Pax6sey sey mice but only when the 30region of Pax6 was intact Kleinjan et al 2001 Schedl et al 1996 Interestingly this 30region contains regulatory elements that are functionally conserved between humans and mice Tyas et al 2006 Further analysis of the full length and truncated YAC transgenes evolutionary sequence comparison and transgenic reporter studies revealed the location and activity of several novel tissue specifi c enhancers Griffi n et al 2002 Kleinjan et al 2006 Additional regulatory elements were identifi ed in a region that was termed distant downstream regu latory region located 130 kb downstream of the Pax6 poly A site regions jeo Fig 1A Griffi n et al 2002 Kleinjan et al 2001 2006 Functional study of this downstream regulatory region proved that it is required for Pax6 expression in some but not all tissues Kleinjan et al 2006 Asidefromtranscriptionalregulation anotheremerging mechamism of gene modulation involves microRNAs Selbach et al 2008 Recently Pax6 was found to be subjected to inhibi tion by miR 450b 5p in the corneal epithelium Shalom Feuerstein et al 2012 As the Pax6 30UTR can be targeted by multiple microRNAs this mode of regulation may allow fi ne tuning of the levels of expression of Pax6 splice variants Taken together the study of Pax6 regulation provides an excel lent paradigm for investigating the regulation of a developmental control gene Kleinjan and van Heyningen