Pax Family Research Articles

The promising role of PAX1 (aliases: HUP48, OFC2) gene methylation in cancer screening.

Background Paired‐box gene 1 (PAX1), a member of the PAX family, plays a role in pattern formation during embryogenesis, and might be essential for development of the vertebral column.Methods PAX1 is silenced by methylation in several cancers and is considered a tumor suppressor gene. Our previous studies reported PAX1 as hypermethylated in cervical cancer tissues, thereby suggesting it as a potential screening marker. Recently, an increasing number of studies have confirmed PAX1 methylation as a promising biomarker in cervical cancer based on its excellent discriminatory ability between high‐grade cervical lesions and normal tissues, resulting in a reduced necessity for referral for colposcopy and biopsy. Additionally, PAX1 is also hypermethylated in other tumors, including those associated with epithelial ovarian cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and endometrial carcinoma, and shows relatively good sensitivity and specificity for the detection of these tumors.ResultsThis review summarizes reports of PAX1 methylation and its promising role in cancer screening, especially that associated with cervical cancer.ConclusionAccording to current evidence, combined testing for human papillomavirus and PAX1 methylation analysis represents an efficacious cervical cancer‐screening protocol.

Abstract 4471: Pax9 regulates squamous cell differentiation and alcohol-associated carcinogenesis in the oro-esophageal epithelium

Abstract Pax9 is a transcription factor of the Pax family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of Pax9 in squamous cell differentiation and carcinogenesis of oro-esophageal epithelium. However, its functional role in differentiation and carcinogenesis remains unclear. In this study, Pax9 deficiency in mouse esophagus promoted cell proliferation, delayed cell differentiation and altered the global gene expression profile. Ethanol exposure down-regulated Pax9 expression in human esophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was down-regulated in human oro-esophageal squamous cell carcinoma (OESCC), and its down-regulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with an isocaloric Lieber-DeCarli liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted NMBA-induced squamous cell carcinogenesis in mouse tongue, esophagus, and forestomach. In conclusion, Pax9 regulates squamous cell differentiation in the oro-esophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. Pax9 down-regulation, at least in part, contributes to alcohol-associated oro-esophageal squamous cell carcinogenesis. Citation Format: Zhaohui Xiong, Shuang Ren, Hao Chen, Yao Liu, Caizhi Huang, Yawan Lyvia Zhang, Joab Otieno Odera, Tong Chen, Ralf Kist, Heiko Peters, Katherine Garman, Zheng Sun, Xiaoxin Luke Chen. Pax9 regulates squamous cell differentiation and alcohol-associated carcinogenesis in the oro-esophageal epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4471.

A novel nuclear localization signal spans the linker of the two DNA-binding subdomains in the conserved paired domain of Pax6.

Paired box (Pax) 6, a member of the Pax family of transcription factors, contains two DNA-binding domains, called the paired domain (PD) and the homeodomain (HD), and plays pivotal roles in development of structures such as the eye, central nervous system and pancreas. Pax6 is a major developmental switching molecule because, for example, ectopic expression of the Pax6 gene can induce ectopic whole eye development. Intensive research has been devoted to elucidating the molecular mechanism(s) involved in the function(s) of Pax6, but many issues remain unexplained. One of the important issues is to identify the nuclear localization signal (NLS) in the PD of Pax6, which is predicted to have a stronger NLS activity than that in the HD. We produced expression plasmid constructs that encode the chick Pax6 protein modified to delete the entire PD except for fragments containing putative NLS sequences, and electroporated them in ovo into the developing chick midbrain to define the NLS of the PD. The results show that the NLS in the PD of chick Pax6 consists of an unusually long sequence of 36 amino acid residues. Within this long NLS motif, the central 18 amino acids comprising two consecutive nine-residue segments showed highest NLS activity; this central area corresponds to the C-terminal half of the third α-helix of the PAI subdomain and the subsequent 11 amino acids of a 16-residue linker between PAI and the adjacent RED subdomain. This information helps to elucidate the molecular mechanism by which Pax6 plays a pivotal role during ontogeny.

The Pax protein Eyegone (Eyg) interacts with the pi-RNA component Aubergine (Aub) and controls egg chamber development in Drosophila

The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Eyg has been shown to be nuclear in the cells where it functions. In this report we describe a new functional cytoplasmic distribution of Eyg during egg chamber development in the female ovarioles. The protein is present from the germarium until stage 10 of cyst development. The majority of egg chambers that develop in the absence of Eyg arrest their development before stage 10, show augmented levels of the telomeric retro-transposon TART-A and low levels of heterochromatin marks in the oocyte nucleus. During the maternal to zygotic transition (MTZ) Eyg seems to play a role in destabilizing germ cell less (gcl) and oo 16 RNA binding protein (orb) mRNAs. We were able to show that Eyg interacts with Aubergine (Aub), a component of the pi-RNA pathway during egg chamber development. This interaction could be essential for Eyg to be retained in the cytoplasm and fulfill its functions there.

Identification of rare paired box 3 variant in strabismus by whole exome sequencing.

To identify the potentially pathogenic gene variants that contributes to the etiology of strabismus. A Chinese pedigree with strabismus was collected and the exomes of two affected individuals were sequenced using the next-generation sequencing technology. The resulting variants from exome sequencing were filtered by subsequent bioinformatics methods and the candidate mutation was verified as heterozygous in the affected proposita and her mother by sanger sequencing. Whole exome sequencing and filtering identified a nonsynonymous mutation c.434G-T transition in paired box 3 (PAX3) in the two affected individuals, which were predicted to be deleterious by more than 4 bioinformatics programs. This altered amino acid residue was located in the conserved PAX domain of PAX3. This gene encodes a member of the PAX family of transcription factors, which play critical roles during fetal development. Mutations in PAX3 were associated with Waardenburg syndrome with strabismus. Our results report that the c.434G-T mutation (p.R145L) in PAX3 may contribute to strabismus, expanding our understanding of the causally relevant genes for this disorder.

Ancestral and novel roles of Pax family genes in mollusks

BackgroundPax genes are transcription factors with significant roles in cell fate specification and tissue differentiation during animal ontogeny. Most information on their tempo-spatial mode of expression is available from well-studied model organisms where the Pax-subfamilies Pax2/5/8, Pax6, and Paxα/β are mainly involved in the development of the central nervous system (CNS), the eyes, and other sensory organs. In certain taxa, Pax2/5/8 seems to be additionally involved in the development of excretion organs. Data on expression patterns in lophotrochozoans, and in particular in mollusks, are very scarce for all the above-mentioned Pax-subfamilies, which hampers reconstruction of their putative ancestral roles in bilaterian animals. Thus, we studied the developmental expression of Pax2/5/8, Pax6, and the lophotrochozoan-specific Paxβ in the worm-shaped mollusk Wirenia argentea, a member of Aplacophora that together with Polyplacophora forms the Aculifera, the proposed sister taxon to all primarily single-shelled mollusks (Conchifera).ResultsAll investigated Pax genes are expressed in the developing cerebral ganglia and in the ventral nerve cords, but not in the lateral nerve cords of the tetraneural nervous system. Additionally, Pax2/5/8 is expressed in epidermal spicule-secreting or associated cells of the larval trunk and in the region of the developing protonephridia. We found no indication for an involvement of the investigated Pax genes in the development of larval or adult sensory organs of Wirenia argentea.ConclusionsPax2/5/8 seems to have a conserved role in the development of the CNS, whereas expression in the spicule-secreting tissues of aplacophorans and polyplacophorans suggests co-option in aculiferan skeletogenesis. The Pax6 expression pattern in Aculifera largely resembles the common bilaterian expression during CNS development. All data available on Paxβ expression argue for a common role in lophotrochozoan neurogenesis.

The Pax gene family: Highlights from cephalopods.

Pax genes play important roles in Metazoan development. Their evolution has been extensively studied but Lophotrochozoa are usually omitted. We addressed the question of Pax paralog diversity in Lophotrochozoa by a thorough review of available databases. The existence of six Pax families (Pax1/9, Pax2/5/8, Pax3/7, Pax4/6, Paxβ, PoxNeuro) was confirmed and the lophotrochozoan Paxβ subfamily was further characterized. Contrary to the pattern reported in chordates, the Pax2/5/8 family is devoid of homeodomain in Lophotrochozoa. Expression patterns of the three main pax classes (pax2/5/8, pax3/7, pax4/6) during Sepia officinalis development showed that Pax roles taken as ancestral and common in metazoans are modified in S. officinalis, most likely due to either the morphological specificities of cephalopods or to their direct development. Some expected expression patterns were missing (e.g. pax6 in the developing retina), and some expressions in unexpected tissues have been found (e.g. pax2/5/8 in dermal tissue and in gills). This study underlines the diversity and functional plasticity of Pax genes and illustrates the difficulty of using probable gene homology as strict indicator of homology between biological structures.

Congenital fusion of cervical vertebrae: a review on embryological etiology

Congenital fusion of cervical vertebrae is a rare anomaly. In this condition, two fused vertebrae appear structurally and functionally as one. This anomaly may be symptomatic or asymptomatic. Myelopathy, limitation in neck movement, muscular atrophy and regional sensory loss are examples of probable morbidity associated with this anomaly. Combination of genetic and environmental factors are involved in pathogenesis of this anomaly. Malformation of notochord, poor performance of retinoids, decreased local blood supply of spine and alteration in genes expression, especially members of Hox and Pax family genes are some of the proposed reasons of congenital fusion of cervical vertebrae. Diagnosis of this congenital anomaly in childhood seems to have an important role in prevention of probable secondary disorders in adulthood. We offer to clinicians that after performing careful physical tests and noticing the presence of signs and symptoms that mentioned in this paper, if a patient suspected to have congenital fusion of cervical vertebrae, genetic tests ought to be performed.

Involvement of pax2 in ovarian development and recrudescence of catfish: a role in steroidogenesis.

PAX2, a member of paired box family, is an essential transcription factor for the organ development in vertebrates including teleosts, yet no evidence has been shown for its involvement in reproduction. To study this, partial- and/or full-length cDNA of pax2 was isolated from the ovary of catfish, Clarias batrachus, along with its other Pax family members, pax1 and pax9 Tissue distribution and ontogeny expression analysis indicated the prevalence of pax2 but not pax1 and pax9 in ovary. Varied phase-wise expression during ovarian cycle and elevation of pax2 after human chorionic gonadotropin induction showed probable regulation by gonadotropins. Pax2 could be localized in various stages of oocytes and in follicular layer of vitellogenic and post-vitellogenic oocytes. To assess the functional significance of pax2, transient RNA silencing was performed using primary catfish ovarian follicle culture, in vitro, and in catfish, in vivo, through ovary-targeted injection of PEI-esiRNA. Pax2 siRNA treatment reduced the expression of various transcripts related to ovarian development like signaling molecules such as wnt4 and wnt5, estrogen receptors, several steroidogenic enzymes and transcription factors. These transitions in transcript levels might have been mediated by Pax2 acting upstream of wnt4/5 that may play a role in steroidogenesis and/or ovarian development along with ad4bp/sf-1 or by direct or indirect interaction with steroidogenic enzyme genes, which is evident from the change in the levels of serum estradiol-17β but not 17α,20β-dihydroxy-4-pregnen-3-one. Taken together, it seems that pax2 has a plausible role during ovarian development and/or recrudescence of catfish either directly or indirectly through Wnt signaling pathway.

GFAP expression is regulated by Pax3 in brain glioma stem cells.

Glioblastomas are understood to evolve from brain glioma stem cells (BGSCs), and yet the biology underlying this model of tumorigenesis is largely unknown. Paired box3 protein (Pax3) is a member of the paired box (Pax) family of transcription factors that is normally expressed during embryonic development, but has recently been implicated in tumorigenesis. The present study demonstrated that Pax3 is differentially expressed in U87MG human glioma cell, BGSC and normal 1800human astrocyte lines. Herein, we identified that the glial fibrillary acidic protein (GFAP), a major intermediate filament protein of mature astrocytes, is directly downregulated during the differentiation of BGSCs via the binding of Pax3 to the promoter region of GFAP. Moreover, siRNA silencing of Pax3 arrested BGSC differentiation, while overexpression of Pax3 promoted the differentiation in BGSCs. Furthermore, we studied the cell proliferation, invasion, apoptosis, differentiation and expression of Pax3 and GFAP in Pax3 siRNA-knockdown and Pax3-overexpressing BGSC models by CCK-8, Transwell migration, flow cytometry and western blot assays. The results indicate that Pax3 regulates GFAP expression, and that Pax3 may contribute to the evolution of BGSCs towards malignancy.

De Novo Assembly and Characterization of Early Embryonic Transcriptome of the Horseshoe Crab Tachypleus tridentatus.

The horseshoe crab Tachypleus tridentatus is a unique marine species and a potential model for marine invertebrate. Limited genomic and transcriptional data are currently available to understand the molecular mechanisms underlying the embryonic development of T. tridentatus. Here, we reported for the first time the de novo transcriptome assembly for T. tridentatus at embryonic developmental stage using Illumina RNA-seq platform. Approximate 38 million reads were obtained and further assembled into 133,212 unigenes. Sequence homology analysis against public databases revealed that 33,796 unigenes could be annotated with gene descriptions. Of the annotated unigenes, we identified a number of key components of several conserved metazoan signaling pathways (Hedgehog, Wnt, TGF-beta and Notch pathways) and other important regulatory genes involved in embryonic development. Targeted searching of Pax family genes which play critical roles in the formation of tissue and organ during embryonic development identified a complete set of Pax family genes. Moreover, the full length T. tridentatus Pax1/9a (TtPax1/9a) and Pax1/9b (TtPax1/9b) cDNA sequences were determined based on the transcriptome, demonstrating the immediate application of our database. Using quantitative real time PCR, we analyzed the expression patterns of TtPax1/9a and TtPax1/9b in different tissues of horseshoe crab. Taking advantage of Drosophila model, we further found that TtPax1/9b, but not TtPax1/9a, can partly rescue the Drosophila homolog Poxm dysfunction-caused lethality at the larval stage. Our study provides the embryonic transcriptome of T. tridentatus which could be immediately used for gene discovery and characterization, functional genomics studies in T. tridentatus. This transcriptome database will also facilitate the investigations of molecular mechanisms underlying embryonic development of T. tridentatus and other marine arthropods as well.

The Diagnostic Utility of PAX8 for Neuroendocrine Tumors

PAX8 is a transcription factor crucial for the development of the kidneys, thyroid glands, and Mullerian system. It is commonly expressed by epithelial tumors from these organs. Recently, the diagnostic utility of PAX8 has been expanded to pancreatic neuroendocrine tumors (NETs). However, PAX8 is not expressed by pancreatic islets during organogenesis. The potential cross-reactivity of polyclonal PAX8 antibody to other PAX family members makes it essential that related findings need to be validated. In this study, we used 4 antibodies (1 polyclonal and 3 monoclonal) on 115 NETs from various organs (pancreas, n=20; thymus, n=16; cervix, n=27; parathyroid, n=7; and thyroid, lung, ileum, stomach, duodenum, appendix, rectum, ovary, and skin, each n=5) to test the utility of PAX8 as a differential diagnosis marker of NETs. Our results showed that NETs from a large variety of organs except for lung and ileum could be immunoreactive to the 2 less specific antibodies that have been observed to cross-react with other PAX proteins (Proteintech polyclonal and Cell Marque MRQ50). However, all NETs were immunonegative for the other 2 monoclonal antibodies specific for the less conserved C-terminal portion of PAX8 (Abcam PAX8R1 and Biocare BC12). We concluded that PAX8 may provide help in the differential diagnosis of NETs in certain specific contexts but the antibody used and the clinical information must be considered.

Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL

Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies. DisclosuresNo relevant conflicts of interest to declare.

Transcriptome analysis of vertebral bone in the flounder, Paralichthys olivaceus (Teleostei, Pleuronectiformes), using Illumina sequencing

The processes underlying vertebral development in teleosts and tetrapods differ markedly in a variety of ways. At present, the molecular basis of teleost vertebral development and growth is poorly understood. Understanding vertebral development at the molecular level is important for aquaculture to prevent vertebral anomalies that can arise from a variety of factors, including excess vitamin A (all-trans retinol, VA) in the diet. To facilitate studies on teloest vertebral development, we performed transcriptome analysis of four month old flounder, Paralichthys olivaceus, vertebrae using next-generation sequencing. Expression profile obtained demonstrates that some members of the hh, bmp, fgf, wnt gene families, and their receptors, hox, pax, sox, dlx and tbx gene families and ntl, which are known to function in notochord and somite development in embryos, are expressed in the vertebrae. It was also showed that in addition to the retinoic acid receptor (Rar), the vertebrae express alcohol dehydrogenase 1 and retinal dehydrogenase 2 which convert VA to all-trans-retinoic acid (RA). The assembled contigs also included cytochrome p450 family members, which inactivate RA, as well as phosphatidylcholine-retinol O-acetyltransferase, which converts VA to all-trans-retinyl ester, a stock form of VA. These data suggest that in teleost vertebrae, expression of various signals and transcription factors which function in the notochord and somite development is maintained until adult stage, and RA metabolism and signaling are active to regulate transcription of RA-responsible genes, such as hedgehog and hox genes. This is the first transcriptome analysis of teleost fish vertebrae.

An emerging role for prdm family genes in dorsoventral patterning of the vertebrate nervous system.

The embryonic vertebrate neural tube is divided along its dorsoventral (DV) axis into eleven molecularly discrete progenitor domains. Each of these domains gives rise to distinct neuronal cell types; the ventral-most six domains contribute to motor circuits, while the five dorsal domains contribute to sensory circuits. Following the initial neurogenesis step, these domains also generate glial cell types—either astrocytes or oligodendrocytes. This DV pattern is initiated by two morphogens—Sonic Hedgehog released from notochord and floor plate and Bone Morphogenetic Protein produced in the roof plate—that act in concentration gradients to induce expression of genes along the DV axis. Subsequently, these DV-restricted genes cooperate to define progenitor domains and to control neuronal cell fate specification and differentiation in each domain. Many genes involved in this process have been identified, but significant gaps remain in our understanding of the underlying genetic program. Here we review recent work identifying members of the Prdm gene family as novel regulators of DV patterning in the neural tube. Many Prdm proteins regulate transcription by controlling histone modifications (either via intrinsic histone methyltransferase activity, or by recruiting histone modifying enzymes). Prdm genes are expressed in spatially restricted domains along the DV axis of the neural tube and play important roles in the specification of progenitor domains, as well as in the subsequent differentiation of motor neurons and various types of interneurons. Strikingly, Prdm proteins appear to function by binding to, and modulating the activity of, other transcription factors (particularly bHLH proteins). The identity of key transcription factors in DV patterning of the neural tube has been elucidated previously (e.g. the nkx, bHLH and pax families), but it now appears that an additional family is also required and that it acts in a potentially novel manner.

Pax2/5/8 and Pax6 alternative splicing events in basal chordates and vertebrates: a focus on paired box domain.

Paired box transcription factors play important role in development and tissue morphogenesis. The number of Pax homologs varies among species studied so far, due to genome and gene duplications that have affected PAX family to a great extent. Based on sequence similarity and functional domains, four Pax classes have been identified in chordates, namely Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6. Numerous splicing events have been reported mainly for Pax2/5/8 and Pax6 genes. Of significant interest are those events that lead to Pax proteins with presumed novel properties, such as altered DNA-binding or transcriptional activity. In the current study, a thorough analysis of Pax2/5/8 splicing events from cephalochordates and vertebrates was performed. We focused more on Pax2/5/8 and Pax6 splicing events in which the paired domain is involved. Three new splicing events were identified in Oryzias latipes, one of which seems to be conserved in Acanthomorphata. Using representatives from deuterostome and protostome phyla, a comparative analysis of the Pax6 exon-intron structure of the paired domain was performed, during an attempt to estimate the time of appearance of the Pax6(5a) mRNA isoform. As shown in our analysis, this splicing event is characteristic of Gnathostomata and is absent in the other chordate subphyla. Moreover, expression pattern of alternative spliced variants was compared between cephalochordates and fish species. In summary, our data indicate expansion of alternative mRNA variants in paired box region of Pax2/5/8 and Pax6 genes during the course of vertebrate evolution.

Phylogenetic analysis and expression patterns of Pax genes in the onychophoran Euperipatoides rowelli reveal a novel bilaterian Pax subfamily

Pax family genes encode a class of transcription factors that regulate various developmental processes. To shed light on the evolutionary history of these genes in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we analyzed the Pax repertoire in the embryonic and adult transcriptomes of the onychophoran Euperipatoides rowelli. Our data revealed homologs of all five major bilaterian Pax subfamilies in this species, including Pax2/5/8, Pax4/6, Pox-neuro, Pax1/9/Pox-meso, and Pax3/7. In addition, we identified a new Pax member, pax-α, which does not fall into any other known Pax subfamily but instead clusters in the heterogenic Pax-α/β clade containing deuterostome, ecdysozoan, and lophotrochozoan gene sequences. These findings suggest that the last common bilaterian ancestor possessed six rather than five Pax genes, which have been retained in the panarthropod lineage. The expression data of Pax orthologs in the onychophoran embryo revealed distinctive patterns, some of which might be related to their ancestral roles in the last common panarthropod ancestor, whereas others might be specific to the onychophoran lineage. The derived roles include, for example, an involvement of pax2/5/8, pox-neuro, and pax3/7 in onychophoran nephridiogenesis, and an additional function of pax2/5/8 in the formation of the ventral and preventral organs. Furthermore, our transcriptomic analyses suggest that at least some Pax genes, including pax6 and pax-α, are expressed in the adult onychophoran head, although the corresponding functions remain to be clarified. The remarkable diversity of the Pax expression patterns highlights the functional and evolutionary plasticity of these genes in panarthropods.

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

Evolution of the vertebrate Pax4/6 class of genes with focus on its novel member, the Pax10 gene.

The members of the paired box (Pax) family regulate key developmental pathways in many metazoans as tissue-specific transcription factors. Vertebrate genomes typically possess nine Pax genes (Pax1–9), which are derived from four proto-Pax genes in the vertebrate ancestor that were later expanded through the so-called two-round (2R) whole-genome duplication. A recent study proposed that pax6a genes of a subset of teleost fishes (namely, acanthopterygians) are remnants of a paralog generated in the 2R genome duplication, to be renamed pax6.3, and reported one more group of vertebrate Pax genes (Pax6.2), most closely related to the Pax4/6 class. We propose to designate this new member Pax10 instead and reconstruct the evolutionary history of the Pax4/6/10 class with solid phylogenetic evidence. Our synteny analysis showed that Pax4, -6, and -10 originated in the 2R genome duplications early in vertebrate evolution. The phylogenetic analyses of relationships between teleost pax6a and other Pax4, -6, and -10 genes, however, do not support the proposed hypothesis of an ancient origin of the acanthopterygian pax6a genes in the 2R genome duplication. Instead, we confirmed the traditional scenario that the acanthopterygian pax6a is derived from the more recent teleost-specific genome duplication. Notably, Pax6 is present in all vertebrates surveyed to date, whereas Pax4 and -10 were lost multiple times in independent vertebrate lineages, likely because of their restricted expression patterns: Among Pax6-positive domains, Pax10 has retained expression in the adult retina alone, which we documented through in situ hybridization and quantitative reverse transcription polymerase chain reaction experiments on zebrafish, Xenopus, and anole lizard.

An Allelic Series at the Paired Box Gene 6 (Pax6) Locus Reveals the Functional Specificity of Pax Genes

The advent of the ocular and nervous system in metazoan evolution coincides with the diversification of a single ancestral paired box (Pax) gene into Pax6, Pax6(5a), and Pax2. To investigate the role of these Pax genes in neural development, we have generated an allelic series of knock-in models at the Pax6 locus. We showed that although Pax6(5a) and Pax2 could not replace Pax6 for its autoregulation in lens induction or for neural differentiation in retina, Pax6(5a) was sufficient for corneal-lenticular detachment. In brain development, cell proliferation in the cerebral cortex and dorsoventral patterning of the telencephalon and neural tube were partially rescued in either knock-in mutant. Contrary to the previous belief, our genetic studies showed that the Pax6 isoform Pax6(5a) could potentially play a role in neuronal differentiation in brain development. Importantly, Pax2 showed greater rescue efficiency than Pax6(5a) in the telencephalon even though the latter was identical to Pax6 outside the paired domain. In studying Ngn2, a Pax6 direct target gene in telencephalon, we showed that the level of Ngn2 expression correlated with the in vitro binding of Pax2, Pax6, and Pax6(5a) paired domain on its enhancer. Our results show that Pax6 is uniquely required for eye development, but in brain development, Pax6 can be functionally substituted by related Pax family genes that share a similar paired domain binding specificity.