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Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors
Yi-Wen Hsieh, Xian-Jie Yang
Neural Development , 2009, DOI: 10.1186/1749-8104-4-32
Abstract: We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates.These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase re-entry, whereas rapid accumulation or reduction of Pax6 protein during the G2/M phase of the cell cycle may be required for specific neuronal fates. These findings thus provide novel insights on the dynamic regulation of Pax6 protein among neurogenic progenitors and the temporal frame of neuronal fate determination.The Pax6 gene encodes an evolutionarily conserved paired homeobox protein critically involved in eye development and retinogenesis [1-5]. In both Drosophila and vertebrates, ectopic expression of Pax6 induces ectopic eyes or
Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis
Joseph A Nielsen, Pierre Lau, Dragan Maric, Jeffery L Barker, Lynn D Hudson
BMC Neuroscience , 2009, DOI: 10.1186/1471-2202-10-98
Abstract: Neuronal progenitors were purified from telencephalic dissociates by a positive-selection strategy featuring surface labeling with tetanus-toxin and cholera-toxin followed by fluorescence-activated cell sorting. Microarray analyses revealed the fractions of miRNAs and mRNAs that were up-regulated or down-regulated in these neuronal progenitors at the beginning of cortical development. Nearly half of the dynamically expressed miRNAs were negatively correlated with the expression of their predicted target mRNAs.These data support a regulatory role for miRNAs during the transition from neuronal progenitors into the earliest differentiating cortical neurons. In addition, by supplying a robust data set in which miRNA and mRNA profiles originate from the same purified cell type, this empirical study may facilitate the development of new algorithms to integrate various "-omics" data sets.Neurogenesis commences in the developing telencephalon when symmetrically dividing neural stem cells in the neuroepithelium begin to divide asymmetrically [1,2]. Neuronal progenitors proliferate and migrate to form the stratified layers of the cortex, with the earliest neurons forming the preplate or primordial plexiform layer [3,4]. A number of genes that are required for the proper formation of the cortex have already been identified including the transcription factors Pax6 and FoxG1 [5,6]. Larger scale genomic approaches have also been used to identify genes important for cortical development, and these studies have added to the catalog of genes that may be required during cortical neurogenesis [7,8].Other participants in gene regulatory networks include microRNAs (miRNAs), which are short non-coding RNA molecules that bind to target mRNAs and cause either RNA degradation or translation inhibition (reviewed in [9,10]). miRNAs were originally identified in the regulation of a developmental transition in C. elegans [11]. miRNAs are expressed in all tissues, but the brain appears to have t
A Large Novel Deletion Downstream of PAX6 Gene in a Chinese Family with Ocular Coloboma  [PDF]
Hong Guo, Limeng Dai, Yanming Huang, Qiong Liao, Yun Bai
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083073
Abstract: Purpose The paired box gene 6 (PAX6) is an essential transcription factor for eye formation. Genetic alterations in PAX6 can lead to various ocular malformations including aniridia. The purpose of this study was to identify genetic defects as the underlying cause of familial ocular coloboma in a large Chinese family. Methods After linkage analysis was carried out in this family, all exons of PAX6 in the proband were sequenced by the Sanger sequencing technique. Then the genome of the proband was evaluated by a microarray-based comparative genomic hybridization (aCGH). Quantitative real-time PCR was applied to verify the abnormal aCGH findings. Results All patients presented bilateral partial coloboma of iris, severe congenital nystagmus, hyperpresbyopia and congenital posterior polar cataracts. Two-point linkage analysis in the autosomal dominant family showed loss of heterozygosity at the D11S914 locus. There was no pathogenic mutation in the exons of PAX6. The aCGH analysis revealed a 681 kb heterozygous deletion on chromosome 11p13. Quantitative real-time PCR verified the deletion in the patients and further confirmed this deletion cosegregation with the ocular coloboma phenotype in the family. Conclusions The 681 kb large deletion of chromosome 11p13 downstream of PAX6 is the genetic cause of the familial ocular coloboma in this large Chinese family. aCGH should be applied if there is a negative result for the mutation detection of PAX6 in patients with ocular coloboma.
Analysis of Area-Specific Expression Patterns of RORbeta, ER81 and Nurr1 mRNAs in Rat Neocortex by Double In Situ Hybridization and Cortical Box Method  [PDF]
Junya Hirokawa, Akiya Watakabe, Sonoko Ohsawa, Tetsuo Yamamori
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003266
Abstract: Background The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space. Principal Findings Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal. Conclusions/Significance The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.
Downstream genes of Pax6 revealed by comprehensive transcriptome profiling in the developing rat hindbrain
Keiko Numayama-Tsuruta, Yoko Arai, Masanori Takahashi, Makiko Sasaki-Hoshino, Nobuo Funatsu, Shun Nakamura, Noriko Osumi
BMC Developmental Biology , 2010, DOI: 10.1186/1471-213x-10-6
Abstract: Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up- or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally.These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.Pax6 is a highly conserved transcription factor that contains two DNA-binding domains, i.e., a paired domain (PD) and a homeodomain. Pax6 has been identified as an essential regulator for the development of the central nervous system (CNS), eyes, nose, pancreas and pituitary gland, mostly through study of the phenotypes of several mouse and rat lines that possess either a spontaneous or an artificial mutation in their Pax6 gene. Although Pax6 heterozygous mutant mice/rats with a semi-dominant mutation are known as Small eye (Sey) mutants and can be bred, their homozygous mutant embryos die soon after birth and exhibit severe phenotypes such as a lack
Pax6 Expression Is Sufficient to Induce a Neurogenic Fate in Glial Progenitors of the Neonatal Subventricular Zone  [PDF]
Eun Sook Jang, James E. Goldman
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0020894
Abstract: Background The forebrain subventricular zone (SVZ) of neonatal mammals contains a large, heterogeneous population of migratory and proliferating precursors of interneurons and glia. These cell types are produced in large numbers in the immediate postnatal period, the glioblasts populating the hemispheres with astrocytes and oligodendrocytes, the neuroblasts migrating to the olfactory bulb to become interneurons. How cell fate decisions are determined or stabilized in this mixed population is not clear, although previous studies indicate the importance of two transcription factors, Pax6 in neurons and Olig2 in glia, and suggest there may be reciprocal repression between these genes. Methodology/Principal Findings In examining the SVZ of neonatal mouse and rat brain, we find that the very large majority of SVZ cells express either Pax6 or Olig2, but few express both. We have used in vivo retro- and lenti-virus injections into the neonatal SVZ and in vitro gene transfer to demonstrate that pax6 over-expression is sufficient to down-regulate olig2 and to promote a neuronal lineage development and migration pattern in olig2-expressing cells. Furthermore, we provide evidence that Pax6 binds to the olig2 promoter and that an HEB sequence in the promoter is required for the Pax6 repression of olig2 transcription. Lastly, we constructed a lentivirus to target olig2-expressing cells in the SVZ to trace their fates, and found that the very large majority developed into glia. Conclusions/Significance We provide evidence for a direct repression of olig2 by Pax6. Since SVZ cells can display developmental plasticity in vitro, the cross-repression promotes a stabilization of cell fates. This repression may be critical in a germinal zone in which immature cells are highly migratory and are not organized into an epithelium.
Stromal derived factor-1 exerts differential regulation on distinct cortical cell populations in vitro
James Pritchett, Clare Wright, Leo Zeef, Bagirathy Nadarajah
BMC Developmental Biology , 2007, DOI: 10.1186/1471-213x-7-31
Abstract: To investigate the role of SDF-1 signaling in the growth and differentiation of cortical cells, we performed numerous in vitro experiments, including gene chip and quantitative RT-PCR analysis. Using SDF-1 medium and AMD3100, a receptor antagonist, we demonstrate that the chemokine signaling regulates key events during early cortical development. First, SDF-1 signaling maintains cortical progenitors in proliferation, possibly through a mechanism involving connexin 43 mediated intercellular coupling. Second, SDF-1 signaling upregulates the differentiation of cortical GABAergic neurons, independent of sonic signaling pathway. Third, SDF-1 enables the elongation and branching of axons of cortical glutamatergic neurons. Finally, cortical cultures derived from CXCR4-/- mutants show a close parallel to AMD3100 treatment with reduced cell proliferation and differentiation of GABAergic neurons.Results from this study show that SDF-1 regulates distinct cortical cell populations in vitro.The cerebral cortex is primarily composed of glutamatergic pyramidal neurons of dorsal telencephalon and GABAergic interneurons that emanate from ventral telencephalon [1]. The generation of cortical neurons is a tightly orchestrated process that involves the commitment of multipotent stem cells to lineage-restricted progenitors, progression of progenitors to postmitotic neurons and their subsequent migration to correct layer positions. The molecular mechanisms that underlie many of these early events are being unraveled. Accordingly, genetic mechanisms involving proneural genes Ngn1/2 and the homeodomain gene Pax6 have been implicated in the specification of pyramidal neurons. Similarly, proneural gene Mash1 and homeodomain genes Dlx 1, 2 that are specific to basal ganglia are known to direct the specification of cortical interneurons [2].In addition to the intrinsic regulators, numerous extrinsic signals that influence the cortical progenitors and neurons have been identified. Among these,
Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma
Mehdi H Shahi, Mohammad Afzal, Subrata Sinha, Charles G Eberhart, Juan A Rey, Xing Fan, Javier S Castresana
BMC Cancer , 2010, DOI: 10.1186/1471-2407-10-614
Abstract: We silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples.Silencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas.Our results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.The Sonic hedgehog (Shh) signaling pathway is crucial for embryonic development and is involved in the fate of many tissues during organogenesis, including the central nervous system [1-4]. Additionally, the Shh signaling pathway has been implicated in stem cell renewal [5] as well as in the development of tumors such as medulloblastoma [1,6], prostate cancer [2,7,8] colorectal carcinoma [9], and glioma [10].This pathway is initiated by ligation of the Shh protein with its receptor PTCH1 on a target cell. Its binding relieves the inhibition of Smoothened (SMO) by PTCH1. The active SMO enters the cytoplasm and activates GLI1. GLI1 is then phosphorylated by the fused ser
ER81 Expression in Breast Cancers and Hyperplasia  [PDF]
YuanYuan Wang,Li Wang,Yue Chen,Lin Li,XuanTao Yang,BaoLin Li,ShuLing Song,LiLin Yang,Yan Hao,JuLun Yang
Pathology Research International , 2011, DOI: 10.4061/2011/980513
Abstract: ER81 is a transcription factor that may contribute to breast cancer; however, little known about the role of ER81 in breast carcinogenesis. To investigate the role of ER81 in breast carcinogenesis, we examined ER81 expression in IDC, DCIS, ADH, HUT, and normal breast tissues by immunohistochemical staining. We found that ER81 overexpression was detected in 25.7% (9/35) of HUT, 41.2% (7/17) of ADH, 54.5% (12/22) of DCIS, and 63.0% (51/81) of IDC. In 20 of breast cancer tissues combined with DCIS, ADH, and HUT, ER81 expression was found in 14/20 (70%) IDC. In these 14 cases all cases were ER81 positive expression in DCIS, 13 of 14 cases were positively expressed of ER81 in ADH and 8 of 14 were positive for ER81 in HUT components. A statistical significance was found between NBT and HUT ( ) and HUT and ADH ( ). Clinical-pathological features analysis of breast cancer revealed that ER81 expression was significantly associated with Her2 amplification and was negatively associated with ER and PR expression. Our results demonstrated that ER81 overexpression was present in the early stage of breast development that suggested that ER81 overexpression may play an important role in breast carcinogenesis. 1. Introduction Breast carcinogenesis is thought to undergo a transition from normal epithelium to invasive carcinoma (IDC) via hyperplasia of usual type (HUT), atypical ductal hyperplasia (ADH), and carcinoma in situ (DCIS) [1]. Over 14% of breast cancer diagnosed in the United States annually are DCIS, and approximately 50% of untreated DCIS will develop into an IDC within 24 years after the original biopsy [2]. However, it is unclear how IDC develop from these lesions. ER81 (ETS-related 81), also called ETS variant 1 (ETV1), is a transcription factor that is a member of the ETS family of DNA-binding proteins [3–5]. Its association with cancer was first noted in Ewing tumors, in which the EWS gene can be translocated onto the ER81 gene and the resultant EWS-ER81 fusion protein exerts oncogenic properties [6]. From then on, many findings suggest that dysregulation of ER81 target genes in disparate tumors like Ewing sarcomas and prostate carcinomas are causally involved in tumorigenesis [7]. Of note, ER81 transcriptional activity is dramatically enhanced upon Her2/Neu (a receptor tyrosine kinase and proto-oncoprotein especially associated with breast cancer) overexpression [8, 9]. Moreover, ER81 mRNA levels are increased in murine cell lines and tumors overexpressing Her2/Neu and also in many human breast cancer cell lines, which suggests that ER81 may contribute
Novel lines of Pax6-/- embryonic stem cells exhibit reduced neurogenic capacity without loss of viability
Jane C Quinn, Michael Molinek, Tomasz J Nowakowski, John O Mason, David J Price
BMC Neuroscience , 2010, DOI: 10.1186/1471-2202-11-26
Abstract: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- ? Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- ? Pax6+/+ chimeras made by embryo aggregation.We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.Pax6 is a highly-conserved transcription factor whose main sites of expression are in the developing eye and central nervous system [1-5]. Homozygous loss-of-function mutations of Pax6 cause failure of eye morphogenesis and severe abnormalities of brain development [6,7]. Pax6 plays an important role promoting neurogenesis; in vivo, loss of Pax6 results in neural progenitors having reduced neurogenic potential [8,9] whereas its over-expression in vitro pushes cells towards a neuronal fate [8,10,11]. The mechanisms by which Pax6 directly promotes neurogenesis are not yet known.Pluripotent embryonic stem (ES) cell lines have provided a means to exploit gene targeting for the analysis of gene function in vivo. In addition, since ES cell lines can be differentiated into a variety of cell types in culture they provide an opportunity to study gene function by comparing the phenotypes of ES-derived cells in vitro. ES cells have been us
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