RESUMO
A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA-seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.
Assuntos
Relógios Circadianos , Ritmo Circadiano/genética , Glândula Pineal , Proteínas de Peixe-Zebra , Animais , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Larva/genética , Larva/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Glândula Pineal/crescimento & desenvolvimento , Glândula Pineal/metabolismo , Glândula Pineal/fisiologia , Análise de Sequência de RNA , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
BACKGROUND: The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pacemaker. Since it is located on the surface of the forebrain, it is accessible for manipulation and, therefore, is a useful model system to analyze pineal gland function and development. We previously analyzed the pineal transcriptome during development and showed that many genes exhibit a highly dynamic expression pattern in the pineal gland. RESULTS: Among genes preferentially expressed in the zebrafish pineal gland, we identified a tissue-specific form of the unc119 gene family, unc119c, which is highly preferentially expressed in the pineal gland during day and night at all stages examined from embryo to adult. When expression of unc119c was inhibited, the formation of the habenular commissure (HC) was specifically compromised. The Unc119c interacting factors Arl3l1 and Arl3l2 as well as Wnt4a also proved indispensible for HC formation. CONCLUSIONS: We suggest that Unc119c, together with Arl3l1/2, plays an important role in modulating Wnt4a production and secretion during HC formation in the forebrain of the zebrafish embryo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Estruturas Animais/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glândula Pineal/embriologia , Proteínas de Peixe-Zebra/biossíntese , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ritmo Circadiano/fisiologia , Especificidade de Órgãos/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
An analysis of rabbit cryopreserved aortic allografts excised on postoperative days (POD) 2, 5, 11, 60, 210, 360, and 720, as well as controls that were untransplanted native aortas and cryopreserved aortas, was performed. On POD2, the number of medial smooth muscle cells in the allografts was reduced to approximately 50%. Ki-67 analysis revealed that medial smooth muscle cells in the allografts proliferated from the 2nd day. By the 11th day, their proliferation ceased and the number of medial smooth muscle cells was restored to almost at the same level as in the controls. Polymorphic microsatellite DNA marker analysis disclosed that the restored medial smooth muscle cells were of donor origin. From 7 months through 2 years, the media of cryopreserved aortic allografts were transformed into acellular structures, in which the elastic fibers were preserved. On the other hand, newly accumulated smooth muscle cells were observed in the adventitia just outside of acellular media after 7 months. In some cases, scattered lamellar calcium deposition was observed in the same regions. This study presents a comprehensive documentation of regeneration and acellular transformation in cryopreserved aortic allografts based on short and long-term analysis.
RESUMO
MOTIVATION: The identification of functional cis-acting DNA regulatory elements is a crucial step towards understanding gene regulation. Ab initio motif detection algorithms have been extensively used in search of regulatory elements. Yet, their success in providing experimentally validated regulatory elements in vertebrates has been limited. RESULTS: Here we report in silico identification and in vivo validation of regulatory elements that determine enhanced gene expression in the pineal gland of zebrafish. Microarray data enabled detection of genes that exhibit high expression in the pineal gland. The promoter regions of these genes were computationally analyzed in order to identify overrepresented motifs. The highest ranking motif identified is a CRX/OTX binding site, known to govern expression in the pineal gland and retina. The second highest ranking motif was not reported before; we experimentally validated its function in vivo by mutational analysis. The methodology presented here may be applicable as a general scheme for finding regulatory elements that contribute to tissue-specific gene expression.
Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica , Glândula Pineal/metabolismo , Elementos Reguladores de Transcrição , Peixe-Zebra/genética , Animais , Regiões Promotoras Genéticas , Peixe-Zebra/metabolismoRESUMO
Pineal function is defined by a set of very narrowly expressed genes that encode proteins required for photoperiodic transduction and rhythmic melatonin secretion. One of these proteins is serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT), which controls the daily rhythm in melatonin production. Here, pineal-specific expression of the zebrafish aanat-2 (zfaanat-2) was studied using in vivo transient expression analyses of promoter-reporter constructs; this revealed that specificity is determined by two regions located 12 kb away from each other. One is the 5'-flanking region, and the other is a 257-bp sequence, located 6 kb downstream of the transcribed region. This 3'-sequence, designated pineal-restrictive downstream module (PRDM), has a dual function: enhancement of pineal expression and inhibition of extrapineal expression. The former is an autonomic property of PRDM whereas the later function requires interaction with the upstream regulatory region of zfaanat-2. Functional analyses of the PRDM sequence revealed that three photoreceptor conserved elements (TAATC) and a single perfect E-box (CACGTG) are crucial for the dual function of PRDM. These results indicate that pineal specificity of zfaanat-2 is determined by the dual functionality of the PRDM and the interaction between upstream regulatory region and downstream photoreceptor conserved elements and E-box element.
Assuntos
Arilalquilamina N-Acetiltransferase/metabolismo , Ritmo Circadiano/fisiologia , Melatonina/metabolismo , Glândula Pineal/enzimologia , Peixe-Zebra/genética , Animais , Arilalquilamina N-Acetiltransferase/genética , Sequência de Bases , Clonagem Molecular , Embrião não Mamífero/enzimologia , Dados de Sequência Molecular , Células Fotorreceptoras/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Peixe-Zebra/embriologiaRESUMO
The zinc finger motif forms a DNA binding domain that is found in a wide variety of proteins. Among them, the members of the zic gene family are highly conserved throughout metazoans. We report here the isolation of two new members of this gene family in zebrafish, zic2.2 and zic5, isolated during random screening for tissue-specific genes. Zic2.2 is closely related to the previously reported zic2 gene, which we propose to rename zic2.1; these two genes form a subfamily with other vertebrate zic2 genes. We compare here the expression patterns of zic2.1, zic2.2, and zic5. All three genes showed dynamic expression patterns starting after the initiation of zygotic transcription, predominantly in the developing neural tube. Compared to zic2.1, zic2.2 was expressed in a similar but distinct manner during early development, particularly in the retina and the forming somites. A zic2.2 ortholog has not been identified in other vertebrate species, suggesting that the zic2.1/zic2.2 pair resulted from a genome duplication event during the evolution of the zebrafish lineage.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/metabolismo , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Somitos/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Dedos de Zinco/genéticaRESUMO
The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pace maker. Here, we have used microarray technology to study the zebrafish pineal transcriptome. Analysis of gene expression at three larval and two adult stages revealed a highly dynamic transcriptional profile, revealing many genes that are highly expressed in the zebrafish pineal gland. Statistical analysis of the data based on Gene Ontology annotation indicates that many transcription factors are highly expressed during larval stages, whereas genes dedicated to phototransduction are preferentially expressed in the adult. Furthermore, several genes were identified that exhibit day/night differences in expression. Among the multiple candidate genes suggested by these data, we note the identification of a tissue-specific form of the unc119 gene with a possible role in pineal development.
Assuntos
Perfilação da Expressão Gênica , Glândula Pineal/metabolismo , Peixe-Zebra/genética , Animais , Encéfalo/metabolismo , Ritmo Circadiano/genética , Análise por Conglomerados , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Fotoperíodo , Glândula Pineal/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Brd4 is a member of the BET (bromodomains and extraterminal) subfamily of bromodomain proteins that includes chromatin-modifying proteins and transcriptional regulators. Brd4 has a role in cell cycle progression, making it indispensable in mouse embryos and cultured cells. The N-terminal domain of Brd4 participates in a fusion oncogene. Brd4 associates with acetylated histones in chromatin, and this association persists during mitosis implicating Brd4 in epigenetic memory. Brd4 sequence, particularly the bromodomains and ET domain, is conserved in the zebrafish and Xenopus laevis proteins reported here. Brd4 is expressed and localized on mitotic chromosomes in early zebrafish embryos before and after the midblastula transition (MBT), indicating that the Brd4-chromosome association is a conserved property that is maintained even before zygotic transcription. The association of Brd4 with acetylated histones may also be conserved in early embryos as we found that histones H3 and H4 are already acetylated during pre-MBT stages.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Mitose , Proteínas de Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Histonas/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Distribuição Tecidual , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Zebrafish is a powerful vertebrate model system for using forward genetics to elucidate mechanisms of early development. We have used chemical mutagenesis to screen for mutants that show defects in the CNS. Here we describe the isolation of the bap28 mutation that leads to abnormalities in the brain starting at midsomitogenesis stages. Mutant embryos display excess apoptosis primarily in the central nervous system (CNS) and die by days 6-7 after fertilization. The mutation was positionally cloned and shown to affect a gene that encodes a large protein with high similarity to the uncharacterized human protein BAP28 and lower similarity to yeast Utp10. Utp10 is a component of a nucleolar U3 small nucleolar RNA-containing RNP complex that is required for transcription of ribosomal DNA and for processing of 18 S rRNA. We show that zebrafish Bap28 likewise is required for rRNA transcription and processing, with a major effect on 18 S rRNA maturation. We suggest that bap28 is required for cell survival in the CNS through its role in rRNA synthesis and processing. Inhibition of p53 protein expression in bap28 mutants led to embryos with morphologically normal appearance, suggesting that p53 is involved in triggering apoptosis in the bap28 mutant CNS. The bap28 mutation provides a genetic approach to study the role of ribosome biogenesis in the development of a vertebrate embryo.
Assuntos
Apoptose/fisiologia , Sistema Nervoso Central/metabolismo , RNA Ribossômico/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mutação , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.
Assuntos
Arilamina N-Acetiltransferase/genética , Regulação da Expressão Gênica no Desenvolvimento , Glândula Pineal/embriologia , Glândula Pineal/fisiologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Genes Reporter/genética , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas/genéticaRESUMO
The Lim-1 gene encodes a LIM-homeodomain transcription factor that is highly conserved among vertebrates and is required for successful gastrulation and head formation. The expression of this gene in the mesoderm of the gastrula is known to require an activin/nodal signal. Earlier studies have shown that the Xenopus Lim-1 (Xlim-1) gene contains an activin response element (ARE) in its first intron, which cooperates with an activin-unresponsive upstream promoter in the regulation of the gene. Here, we show that the Xlim-1 ARE contains a cluster of FAST-1/FoxH1 and Smad4 recognition sites; such sites have been shown to mediate activin/nodal responses in other genes. By using reporter constructs with mutated FAST-1/FoxH1 sites and FAST-1/FoxH1 protein chimeras, we show that the regulation of Xlim-1 by activin depends on FAST-1/FoxH1 function. Comparative studies on the zebrafish lim1 gene indicate the presence of FoxH1 sites in the first intron of this gene and provide evidence for the requirement for FoxH1 function in its regulation. These results illuminate the conserved nature of the transcriptional regulation of the Lim-1 gene in different vertebrate animals.