Reiko Toyama

LIM domains: multiple roles as adapters and functional modifiers in protein interactions

The LIM domain is a specialized double-zinc finger motif found in a variety of proteins, in association with domains of divergent functions or forming proteins composed primarily of LIM domains. LIM domains interact specifically with other LIM domains and with many different protein domains. LIM domains are thought to function as protein interaction modules, mediating specific contacts between members of functional complexes and modulating the activity of some of the constituent proteins. Nucleic acid binding by LIM domains, while suggested by structural considerations, remains an unproven possibility. LIM-domain proteins can be nuclear, cytoplasmic, or can shuttle between compartments. Several important LIM proteins are associated with the cytoskeleton, having a role in adhesion-plaque and actin-microfilament organization. Among nuclear LIM proteins, the LIM homeodomain proteins form a major subfamily with important functions in cell lineage determination and pattern formation during animal development.

Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

ZEBRAFISH SEROTONIN N-ACETYLTRANSFERASE-2 : MARKER FOR DEVELOPMENT OF PINEAL PHOTORECEPTORS AND CIRCADIAN CLOCK FUNCTION

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish lar...

Functional development of the zebrafish pineal gland: light-induced expression of period2 is required for onset of the circadian clock.

In zebrafish, the pineal gland is a photoreceptive organ that contains an intrinsic circadian oscillator and exhibits rhythmic arylalkylamine‐N‐acetyltransferase (zfaanat2) mRNA expression. In the present study, we investigated the role of light and of a clock gene, zperiod2 (zper2), in the development of this rhythm. Analysis of zfaanat2 mRNA expression in the pineal gland of 3‐day‐old zebrafish embryos after exposure to different photoperiodic regimes indicated that light is required for proper development of the circadian clock‐controlled rhythmic expression of zfaanat2, and that a 1‐h light pulse is sufficient to initiate this rhythm. Analysis of zper2 mRNA expression in zebrafish embryos exposed to different photoperiodic regimes indicated that zper2 expression is transiently up‐regulated by light but is not regulated by the circadian oscillator. To establish the association between light‐induced zper2 expression and light‐induced clock‐controlled zfaanat2 rhythm, zPer2 knock‐down experiments were performed. The zfaanat2 mRNA rhythm, induced by a 1‐h light pulse, was abolished in zPer2 knock‐down embryos. These experiments indicated that light‐induced zper2 expression is crucial for establishment of the clock‐controlled zfaanat2 rhythm in the zebrafish pineal gland.

Perturbation of rRNA Synthesis in the bap28 Mutation Leads to Apoptosis Mediated by p53 in the Zebrafish Central Nervous System

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.

LIM6, A NOVEL LIM HOMEOBOX GENE IN THE ZEBRAFISH : COMPARISON OF ITS EXPRESSION PATTERN WITH LIM1

A novel LIM class homeobox gene, lim6, was isolated from a zebrafish embryonic cDNA library. The encoded protein shares a high degree of sequence similarity with the previously described Lim1 and Lim5 proteins. This study compares the spatial and temporal expression pattern of the closely related lim6 and lim1 genes during early embryogenesis. Generally, lim6 mRNA was found at rather low amounts compared to lim1 mRNA. At the shield stage, lim6 mRNA, similar to lim1 mRNA, was predominantly expressed in the shield. Lim6 was transiently expressed in a restricted region of the anterior neural plate at the bud stage, distinct from the expression of lim1 in the notochord and the pronephros and pronephric ducts. During the segmentation period, the lim6 gene started to be expressed in single cells in the spinal cord, followed by a gradually increasing wide‐spread expression throughout the CNS. During this stage, lim1 mRNA disappeared in the notochord and pronephric ducts and was found in the pronephroi and single cells in the CNS. In 24 hr embryos, lim6 and lim1 were expressed in the fore‐, mid‐, and hindbrain and the spinal cord, except that lim1 mRNA was limited to two small domains in the telencephalon, whereas lim6 mRNA was widely expressed in this region. A comparison of expression of lim1and lim6 and of the previously characterized lim5 show that, in spite of close sequence similarity, distinct expression patterns imply nonredundant functions for each member of this group of genes. Dev. Dyn. 209:406–417, 1997.

Pineal‐specific expression of green fluorescent protein under the control of the serotonin‐N‐acetyltransferase gene regulatory regions in transgenic zebrafish

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.

Systematic identification of rhythmic genes reveals camk1gb as a new element in the circadian clockwork.

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.

Regulation of the Lim-1 Gene Is Mediated Through Conserved FAST-1/FoxH1 Sites in the First Intron

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.

Transcriptome analysis of the zebrafish pineal gland.

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. Developmental Dynamics 238:1813–1826, 2009.