Transcriptome Analysis of Ciona intestinalis to Identify Genes Preferentially Expressed in the Larval Brain
ABSTRACT: The larval brain of Ciona intestinalis has similar architecture to that of vertebrates, but is only composed of approximately 330 cells. Transgenic embryos that carried Ci-beta-tubulin(promoter)::Kaede exhibited robust Kaede expression in the larval brain. Kaede-expressing cells were isolated, and their transcriptome was compared with that of cells that did not express Kaede using an oligonucleotide-based microarray. Our analysis identified 565 candidate genes that were preferentially expressed in the larval brain, 77 of which have previously been reported to be brain-related. The 565 genes included transcription factors, such as Otx, en, Pax3/7, Prop-A, Lhx1, Six3/6, Unc4-A, FoxC, and DMRT1; and signal transduction molecules, such as FGF4/5/6, Hedgehog1, Hedgehog2, patched, Fringe1, and Dkk3. Nearly 30 of the identified genes coded for receptors for neurotransmitters, neuropeptides or hormone pepetides. In addition, 15 genes encoded neuropeptides and hormone peptides, five of which were novel. Our catalog of genes that are expressed in the Ciona larval brain provides a foundation for future studies exploring the complex gene regulatory networks that mediate chordate brain development and function. Overall design: Two samples (Brain vs Cells without Brain),Two biological replicates,Dye Swap design
Project description:The larval brain of Ciona intestinalis has similar architecture to that of vertebrates, but is only composed of approximately 330 cells. Transgenic embryos that carried Ci-beta-tubulin(promoter)::Kaede exhibited robust Kaede expression in the larval brain. Kaede-expressing cells were isolated, and their transcriptome was compared with that of cells that did not express Kaede using an oligonucleotide-based microarray. Our analysis identified 565 candidate genes that were preferentially expressed in the larval brain, 77 of which have previously been reported to be brain-related. The 565 genes included transcription factors, such as Otx, en, Pax3/7, Prop-A, Lhx1, Six3/6, Unc4-A, FoxC, and DMRT1; and signal transduction molecules, such as FGF4/5/6, Hedgehog1, Hedgehog2, patched, Fringe1, and Dkk3. Nearly 30 of the identified genes coded for receptors for neurotransmitters, neuropeptides or hormone pepetides. In addition, 15 genes encoded neuropeptides and hormone peptides, five of which were novel. Our catalog of genes that are expressed in the Ciona larval brain provides a foundation for future studies exploring the complex gene regulatory networks that mediate chordate brain development and function. Two samples (Brain vs Cells without Brain),Two biological replicates,Dye Swap design
Project description:The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain. Overall design: Single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. Bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression.
Project description:The number of newly-formed neurons declines rapidly during aging. Here we describe an important mechanism that contributes to this decline via Wip1-dependent regulation of neuronal differentiation. We found that Wip1 is expressed in neural stem/progenitor cells (NPCs) of the mouse subventricular zone and its upregulation at physiological levels maintained higher NPC numbers and neuronal differentiation in old mice. This resulted in markedly improved neuron formation and rescued a functional defect in fine odor discrimination in old mice. We identified Dkk3 as a key downstream target of Wip1 and found that its expression in SVZ is restricted to NPCs. Functionally, Dkk3 inhibited neuroblast formation by suppressing Wnt signaling, while deletion of Dkk3 or pharmacological reactivation of the Wnt pathway improved neuron formation and olfactory function in aged mice. We propose that Wip1 controls a Dkk3-dependent inhibition of neuronal differentiation during aging and thus regulating Wip1 levels could prevent certain aspects of functional decline of the aging brain. We found if neurospheres were derived from 18 months old mice, Wip1 transgenic neurospheres were more neurogenic than wt ones. This microarray was a pilot experiment to search the mechanism how Wip1 Transgene promoted neurogenesis, and found Dkk3 as a potential mediator. WT vs Wip1Tg neurospheres were cultured from mouse brain, and gene expression was compared using Illumina mouseWG-6 array
Project description:In Apis mellifera, the female eggs can develop into workers or queen depending on the diet offered during early development. The outputs of the developed honeybee females are two morphs with particular morphological traits and related physiology. The differential feeding regime experienced by the queen and the worker larvae of the honeybee Apis mellifera shapes a complex endocrine response cascade that ultimately sets up differences in brain morphologies. Herein we report on aspects of brain morphogenesis during larval development and the brain gene expression signature of fourth instar larvae (L4) of both castes, a developmental stage characterized by the greatest differences in juvenile hormone (JH) titers between castes Using results from the hybridization of whole genome-based oligonucleotide arrays with RNA samples from brain of fourth instar larvae honeybees of both castes we present a list of differentially expressed genes. Analysis used one dye-swap combination to compare workers and queens brain development at fourth instar larvae when juvenile hormone titers is higher in queens.
Project description:Hox cluster genes play crucial roles in the establishment of the body plan along the antero-posterior axis during animal development. Hox genes are expressed in the chordate endodermal tissues, and unraveling functions there is necessary for elucidating the mechanisms of endoderm specification. In the invertebrate chordate Ciona intestinalis, the endodermal tissues are in the premature state at the larval stage, and they form the differentiated digestive tract during metamorphosis. In this study, we showed that a Hox gene Ci-Hox10 is required for the formation of the intestine during metamorphosis. To know the downstream genes that are controlled by Hox10, microarray analysis of Hox10 knock-down embryos was performed. Gene expressions in the tail region of Hox10 knockdown embryos that were injected with Hox10 morpholino (MO) and control embryos of Ciona intestinalis were examined by comparative analysis with two color detection. Dye-swap analysis was carried out.
Project description:Few families of signaling factors have been implicated in the control of development. Here we identify the neuropeptides nociceptin and somatostatin, a neurotransmitter and neuroendocrine hormone, as a class of developmental signals in chick and zebrafish. We show that signals from the anterior mesendoderm are required for the formation of anterior placode progenitors with one of the signals being somatostatin. Somatostatin controls ectodermal expression of nociceptin and both peptides regulate Pax6 in lens and olfactory progenitors. Consequently, loss of somatostatin and nociceptin signaling leads to severe reduction of lens formation. Our findings not only uncover these neuropeptides as developmental signals, but also identify a long-sought-after mechanism that initiates Pax6 in placode progenitors and may explain the ancient evolutionary origin of neuropeptides, pre-dating a complex nervous system. We used progenitors for anterior and posterior sensory placodes dissected from chick embryos HH5-7; these were either processed immediately or cultured for 5 hrs to hybridise to Affymetrix chick array. We aimed to identify genes that are co regualted with Pax6, a key regulator of lens and olfactory progenitor cells. Pax6 is normally present in anterior, but not posterior placode precursors, but upregulated in both after 5 hrs culture.
Project description:Medulloblastoma (MB) is one of the most common malignant pediatric brain tumor. The aim of our study was to understand which mechanisms are involved in aberrant activation of Wnt pathway and its association with MB tumor development. We studied the expression profiles of 19 MBs tumors using a genome-wide approach, furthermore we have included the data obtained from four independent public Datasets of gene expression (see README file below) to increase the statistical power of the study. The expression profiles identified the Wnt inhibitor gene, DKK3, significant down-regulated. DKK3 down-regulation was confirmed by qPCR in 80% of samples (27/33 MB tumors and in 3/5 MB human cell lines). Since DKK3 was found methylated in several cancers we also investigated the status of methylated CpG sites within the three promoters by a combination of two approaches: the MSP and pyrosequencing. MSP showed no methylation in all promoters while the pyrosequencing, a quantitative technique, indicated a very low level of methylation. To verify the epigenetic involvement on DKK3 silencing, we also studied the effect of Trichostatin A (TSA) in 4 MB cell lines (DAOY; D283; D425; D458). After TSA treatment we observed a significant DKK3 mRNA expression increase in 3/4 cell lines compared to the no treated cells. DAOY cells, that have a normal DKK3 expression value, did not change significantly the DKK3 expression after TSA treatment. To further validate the role of histone tail modifications as an epigenetic silencing mechanism for DKK3 in MB, we performed Chromatin Immunoprecipitation (ChIP) experiment using antibodies against acetylated histones. According to our earlier results, TSA treatment increased the signal of the histone modification meH3K4 which is related to a transcriptionally active gene respect to the untreated controls. Finally, we evaluated the DKK3 protein in three MB cell lines (DAOY; D283; D425) after 24h of TSA treatment, by immunofluorescence. We analysed 19 Medulloblastoma tumor from pediatric patients, a commercial pool (Clontech) of 24 normal cerebellum from female and male Caucasians (ages: 16-70 years; cause of death: sudden death) and a pool of 10 normal cerebellum from children (6 Caucasians and 4 African Americans; ages: 0-16 years; cause of death: sudden death) using 44K whole genome oligonucleotids microarray
Project description:The goal of this study is to investigate the molecular mechanism of lhx1 on regulation of pronephros formation during the early embryonic development. In the vertebrate embryo the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. The animal cap cells can be induced by treatment of activin and retinoic acid to differentiate into pronephros tissue. In this study we investigated the role of Lhx1 in differentiation of pronephros by depleting lhx1 in the organ culture system. We generated the gene expression profile of early pronephros tissue, and demonstrated that expression of genes from all the kidney domains is affected by the absence of lhx1. Taken together our results highlight an essential role for Lhx1 in pronephros formation. lhx1 is involved in driving specification of intermediate mesoderm into nephrogenic mesenchyme. Lhx1 is initially expressed throughout the entire intermediate mesoderm. To determine the role of lhx1 pronephros formation, we performed a microarray analysis using an explant culture system. Xenopus tissue explants can be surgically isolated and cultured under specific conditions to be driven towards many distinct tissue types. Formation of pronephric cell fates is induced by culturing isolated explants in the presence of Activin and RA (AcRA). Treatment of dissected explants of stage 9 blastulae embryos with 10ng/ml Activin and 1x10-4 M retinoic acid can induce differentiation of the pluripotent ectoderm into pan-kidney tissue. For this experiment, both blastomeres of 2-cell embryos were injected with a total of 800pg lhx1 DEED-AS. Explants were dissected and treated with AcRA and expression of pax8 at stage 15 (based on timing of paired control whole embryos) was analyzed. We observed a lack of induction of pax8 expression in lhx1-depleted explants under AcRA treatment conditions in which expression of this gene is normally induced. Based on this observation, microarray analysis was carried out to identify genes whose expression is affected by the absence of lhx1. Explants of injected embryos with 800pg of lhx1 DEED-AS were dissected, treated with pronephric tissue inductive conditions (AcRA) and harvested after 24 hours incubation at 14C (Fig. S5B). The sibling control embryos reached stage 12.5. Explants from uninjected embryos +AcRA and -AcRA as well as explants from DEED injected embryos -AcRA were also harvested. Approximately 12 caps were pooled for each RNA preparation and the analysis was performed using triplicates.
Project description:Expression profiling of wild-type and Lhx1 null mouse definitive endoderm cultures using Illumina whole genome mouse V2 arrays. The hypothesis tested was that Lhx1 regulates gene expression of definitive endoderm and anterior mesendoderm genes Overall design: Lhx1 null and littermate control wild-type embryonic stem cells were generated from blastocyst outgrowths. Total RNA was obtained from four wild-type ES cell clones and four Lhx1 null ES cell clones differenitated for five and six days with Activin-A to enrich for defintive endoderm cells and hybridized to Illumina WG6_V2 arrays