Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

Transgene expression in zebrafish: A comparison of retroviral-vector and DNA-injection approaches.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-alpha enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.

NIEHS/EPA Workshops. Growth and differentiation factors.

The work group identified a number of research areas where they felt there were significant data gaps where additional research was critical to better under standing of the origins of birth defects and to developing ways for their prevention. These included: 1. Studies designed to determine the role of growth and differentiation factors during pre- and postimplantation stages of development. These investigations could include descriptive studies involving the localization and timing of genes expressed and their products, but must also emphasize and include studies involving the function of these molecules and their interactions in normal and abnormal development. 2. Studies designed to develop and utilize models for investigating normal and abnormal development. These approaches could include in vivo and in vitro techniques, such as creation of genetically defined systems and cell, organ, and whole embryo cultures. These technologies should emphasize ways to study the functions of growth and differentiation factors and the effects of environmental factors. 3. Studies designed to identify environmental agents and their targets in embryonic, extraembryonic, and maternal tissues that may play a role in producing developmental abnormalities through peturbations of growth and differentiation factors. 4. Studies designed to determine cellular and molecular mechanisms for protection and recovery from environmental insults.

Retinoic acid receptor alpha function in vertebrate limb skeletogenesis: a modulator of chondrogenesis.

Retinoic acid is a signaling molecule involved in the regulation of growth and morphogenesis during development. There are three types of nuclear receptors for all-trans retinoic acid in mammals, RAR alpha, RAR beta, and RAR gamma, which transduce the retinoic acid signal by inducing or repressing the transcription of target genes (Leid, M., P. Kastner, and P. Chambon. 1992. Trends Biochem. Sci. 17:427-433). While RAR alpha, RAR beta, and RAR gamma are expressed in distinct but overlapping patterns in the developing mouse limb, their exact role in limb development remains unclear. To better understand the role of retinoic acid receptors in mammalian limb development, we have ectopically expressed a modified RAR alpha with constitutive activity (Balkan, W., G.K. Klintworth, C.B. Bock, and E. Linney. 1992. Dev. Biol. 151:622-625) in the limbs of transgenic mice. Overexpression of the transgene was associated with marked pre- and postaxial limb defects, particularly in the hind limb, where expression of the transgene was consistently seen across the whole anteroposterior axis. The defects displayed in these mice recapitulate, to a large degree, many of the congenital limb malformations observed in the fetuses of dams administered high doses of retinoic acid (Kochhar, D.M. 1973. Teratology. 7:289-295). Further analysis of these transgenic animals showed that the defect in skeletogenesis resided at the level of chondrogenesis. Comparison of the expression of the transgene relative to that of endogenous RAR alpha revealed that downregulation of RAR alpha is important in allowing the chondrogenic phenotype to be expressed. These results demonstrate a specific function for RARalpha in limb development and the regulation of chondroblast differentiation.

Magnetic resonance microscopy of embryos.

We demonstrate that magnetic resonance (MR) microscopy provides a mechanism to investigate normal and abnormal developmental anatomy in a non-destructive and distortion-free manner. Techniques for the fixation, embedding, perfusion and image acquisition of embryos between 3 and 30 mm crown rump length are described. We describe the perfusion of a contrast agent to enhance images of the developing embryonic vasculature. Data are acquired as three-dimensional isotropic arrays which permit images to be reformatted retrospectively in any plane. The data are available for archiving, distributing and for post-acquisition manipulations. MR microscopy is a fast technique for producing three-dimensional reconstructions and is free from registration and sectioning artifacts.

Retinoid signaling and the generation of regional and cellular diversity in the embryonic mouse spinal cord.

Retinoid-dependent gene expression accompanies the emergence of distinct regions and cell classes in the mouse spinal cord around midgestation. We asked whether changes in the expression of retinoid signaling molecules and retinoid-responsive genes reflect the establishment of this regional and cellular diversity. At E10.5, retinoic acid (RA) receptors (RAR)alpha, RAR beta, the retinoid X receptor (RXR) gamma, cellular RA binding protein (CRABP)I, CRAPBII, and cellular retinol binding protein (CRBP)I mRNAs are found throughout the entire anterior-posterior (AP) axis of the cord, as is RA (Colbert et al. [1993] Proc. Natl. Acad. Sci. U.S.A. 90:6572-6576) and RA-sensitive transgene expression (Balkan et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:3347-3351). At E12.5, RA, transgene expression, and RAR beta become restricted to the cervical and lumbar cord. RAR alpha, CRABPI, and RXR gamma, however, are found throughout the AP extent. CRABPII and CRBPI, although expanded within the cervical and lumbar regions, are also found throughout the AP axis. Thus, several retinoid signaling molecules continue to be expressed beyond distinct regions of the spinal cord where RA is available and some RA-responsive genes are either restricted or enhanced. Exogenous RA can activate a more widespread response resulting in ectopic transgene and RAR beta expression in the thoracic and sacral cord. Not all RA-sensitive genes, however, respond; CRABPII and CRBPI expression patterns are unchanged. Finally, not every cell within the normal or exogenously induced domains of RA-dependent gene expression responds to RA, nor does every cell express RA receptors or binding proteins. Thus, regional and cellular differences in the distribution of the known retinoid receptors and binding proteins do not predict absolutely where or whether retinoid sensitive genes will be expressed or where retinoids will be available in the developing spinal cord. Instead, retinoid-mediated gene expression in the cervical and lumbar cord seems to reflect retinoid responses that rely both on the local availability of retinoids, the identity of the responding gene, and an indeterminate array of retinoid signaling molecules.

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector.

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos.

Fibroblasts that express aromatic amino acid decarboxylase have increased sensitivity to the synergistic cytotoxicity of L-dopa and manganese.

Manganism, a neurodegenerative disease that can follow chronic exposure to Mn, has been associated with lesions in the basal ganglia and depletion of dopamine and its metabolites in this brain region. Herein, we have tested the hypothesis that oxidation of catechols is a critical component of Mn-induced cytotoxicity. To eliminate confounding metabolic pathways, a nonneuronal cell line, Chinese hamster ovary (CHO) fibroblasts, was transfected with a cDNA for bovine aromatic amino acid decarboxylase, and a high expressing clone was isolated (CHO/AADC). Exposure of wild-type (CHO/WT) or CHO/AADC cultures to L-dopa (62 to 500 microM) resulted in intracellular accumulation of L-dopa or L-dopa and dopamine, respectively, that was concentration-dependent. Intracellular catechol levels in CHO/AADC cells were double those in CHO/WT cultures. No dopac was identified intra- or extracellularly. Addition of MnCl2 (125 to 500 microM) resulted in cytotoxicity that progressed with increasing concentrations of L-dopa or Mn. Neither L-dopa nor MnCl2 alone was toxic at these concentrations, and cytotoxicity was completely abrogated by substitution of L-tyrosine for L-dopa. Although CHO/AADC cultures were more sensitive than CHO/WT to L-dopa and Mn, this was completely accounted for by the differences in intracellular catechol levels between the two cell lines. Preformed melanin or dopac were low-potency cytotoxins only at high MnCl2 concentrations. These results indicate that Mn and intracellular L-dopa and dopamine, but not extracellular dopac or melanin, are potent synergistic cytotoxins.

Magnetic resonance microscopy of mouse embryos.

The increased use of the mouse as a model for various aspects of mammalian biology has caused a renewed interest in developing strategies for examining and comparing normal and abnormal mouse embryonic development and anatomy. In this study, we have explored the use of magnetic resonance microscopy as a tool for these purposes. Techniques for the fixation, embedding, perfusion, and image acquisition of mouse embryos are described. The perfusion of bovine serum albumin-diethylenetriamine pentaacetic anhydride-gadolinium as a contrast agent enhances images of the developing embryonic vasculature during critical stages of organogenesis and allows for comparisons when embryos have been treated with teratogens such as retinoic acid. The acquired three-dimensional data sets are available for archiving, distributing, and postacquisition manipulations such as computer segmentation of anatomical structures.

Constitutively active retinoid receptors exhibit interfamily and intrafamily promoter specificity.

Retinoid receptors are ligand activated transcription factors that regulate gene transcription through a complex network of interactions with members of the nuclear hormone receptor superfamily. Although ligand is required for trans-activation, addition of ligand to mammalian cells in vitro complicates the study of individual activated retinoid receptors. In order to circumvent this problem we have constructed a series of retinoid receptors which do not require ligand for trans-activation. This was accomplished by fusing the acidic activation domain of the herpes simplex viral protein VP16 to the carboxyl terminus of individual retinoid receptors. All of the chimeric receptors were found to exhibit constitutive trans-activation activity in CV-1 and P19 cells when cotransfected with a reporter that contained a trimerized retinoic acid receptor-beta 2 (RAR beta 2) retinoic acid response element. Further analysis conducted on reporters containing either the RAR beta 2 promoter or the rat cellular retinol binding protein II (rCRBPII) promoter showed that promoter specificity was well conserved between the chimeric receptors in the absence of exogenous retinoid and their ligand-induced native counterparts. Moreover, on the RAR beta 2 promoter reporter construct, the chimeric retinoid receptors displayed both cell type and inter- and intrafamily differences in trans-activation, whereas, trans-activation of the rCRBPII in the absence of exogenous ligand in CV-1 and P19 cells was found to be stimulated only by chimeric retinoid X receptor-alpha (RXR alpha). In P19 cells trans-activation of the rCRBPII promoter by RXR alpha v in the absence of exogenous ligand was inhibited by RAR alpha and the constitutive forms of RAR alpha, RAR beta, RAR gamma, RXR beta, and to a lesser extent RXR gamma.

Local sources of retinoic acid coincide with retinoid-mediated transgene activity during embryonic development.

We have assessed whether retinoic acid (RA) comes from local sources or is available widely to activate gene expression in embryos. We used an RA-responsive indicator cell line, L-C2A5, to localize RA sources. In these cells, an RA-sensitive promoter/lacZ reporter construct used previously by us to produce indicator transgenic mice is induced globally by RA in medium or locally by RA released at physiological concentrations (1 nM) from AG-1X2 resin beads. Furthermore, the cells are differentially responsive to the 9-cis and all-trans isomers of RA at low concentrations. Indicator transgenic mice with the same promoter/reporter construct were used to identify regions of RA-mediated gene activation. There are distinct domains of lacZ expression in the cervical and lumbar spinal cords of embryonic indicator mice. This pattern might reflect localized RA sources or restricted spatial and temporal expression of RA receptors, binding proteins, or other factors. To resolve this issue we compared the pattern of transgene activation in indicator cell monolayers cocultured with normal embryonic spinal cords with that in transgenic spinal cords. The explants induced reporter gene expression in L-C2A5 monolayers in a pattern identical to that in transgenic mice: alar regions of the cervical and lumbar cord were positive whereas those in the thoracic and sacral regions were not. We conclude that restricted sources of RA in the developing spinal cord mediate the local activation of RA-inducible genes. Thus, region-specific gene activation in embryos can be mediated by precisely localized sources of inductive molecules like RA.

Retinoic acid induction and regional differentiation prefigure olfactory pathway formation in the mammalian forebrain.

We have used an in vitro assay to identify sources of retinoic acid (RA) and transgenic mice to identify target domains in the developing forebrain. RA participates in a sequence of events that leads to the establishment of the olfactory pathway. First, the lateral cranial mesoderm activates an RA-inducible transgene in neuroepithelial cells in the olfactory placode and the ventrolateral forebrain. Then, neurons and neurites begin to differentiate in these two regions. Finally, olfactory axons grow specifically into the ventrolateral forebrain and subsequently are limited to the olfactory bulb rudiment. The coordination of these events, perhaps by common signals, implies that retinoid induction and retinoid-activated region-specific transcriptional regulation may help to define a forebrain subdivision and the peripheral neurons that provide its primary innervation.

Mapping of the mouse Rar loci encoding retinoic acid receptors RAR alpha, RAR beta and RAR gamma.

Nuclear retinoic acid receptors RAR alpha, RAR beta and RAR gamma are transcription factors that bind all-trans retinoic acid as their ligand and mediate its action by activating particular set of genes that contain retinoic acid responsive elements in their promoter-enhancers. We have mapped genetic loci for these genes using restriction fragment length variants (RFLVs) in interspecific backcross mice. None of the Rar loci cosegregated with each other or with the new subclass of retinoid receptors, Rxr loci. Rara mapped to mChr 11, Rarb mapped to mChr 14, and Rarg mapped to mChr 15. The results are consistent with the previous reports and the human data in terms of syntenic homology between mouse and human chromosomes.

The mouse retinoid-X receptor-gamma gene: genomic organization and evidence for functional isoforms.

Retinoid-X receptors (RXRs) are a subfamily of the steroid/thyroid nuclear receptor superfamily. There are three RXR genes: alpha, beta, and gamma, each with a distinct expression pattern and chromosomal location. In this study we describe the genomic organization of the mouse RXR gamma gene, identify a second mouse (m) RXR gamma isoform, and map the exons of the two mRXR gamma isoforms on the gene. The new mRXR gamma isoform, called gamma 2, has a unique 5'-untranslated region, and is expressed highly in both cardiac and skeletal muscles, but very little in the liver, while the mRXR gamma 1 is expressed in the brain and muscle. The gamma 2 isoform is also a functional receptor and can transactivate the retinoid response elements of the apolipoprotein-AI and cellular retinol-binding protein-II genes. The mRXR gamma gene spans at least 50 kilobases of DNA on mouse chromosome 1 and has nine introns ranging from 107 basepairs to more than 30 kilobases. From the genomic separation of the two unique 5'-ends of the mRXR gamma isoforms, it is suggested that this gene may have two promoters, similar to those described for the three RAR genes. The results of this study along with those of previously published studies suggest that there may be more than two isoforms of mRXR gamma.

Mapping of the mouse Rxr loci encoding nuclear retinoid X receptors RXR alpha, RXR beta, and RXR gamma.

Recently, a novel subgroup of nuclear hormone receptors called RXRs implicated for retinoid-mediated gene regulation have been identified. RXRs appear to interact with many other nuclear hormone receptors and modulate their functions. We have mapped genetic loci Rxra, Rxrb, and Rxrg encoding three RXR subtypes, RXR alpha, RXR beta, and RXR gamma, respectively, using interspecific backcross mice. None of the Rxr loci cosegregated with each other or with the retinoic acid receptor loci (Rar) mapped previously. Rxra mapped to Chr 2 near the centromere, Rxrb mapped to the H-2 region of Chr 17, and Rxrg was tightly linked to the Pbx gene on distal Chr 1. These results underscore that RXR genes are dispersed in the genome.

Parent-specific expression of a human keratin 18/beta-galactosidase fusion gene in transgenic mice.

Insertion of a human keratin 18 (K18)-bacterial beta-galactosidase (LacZ) fusion gene into mice has led to a unique transgenic line in which expression of the transgene is subject to unusual germ line-specific, genomic imprinting effects. Fetal expression of the LacZ reporter gene depends on the gender of the transmitting parent, with appropriate expression in liver after maternal inheritance, and ectopic expression in retina and mesodermal tissues after paternal inheritance. This tissue-specific imprinting pattern is superimposed upon a basic expression pattern which is unaffected by parental inheritance. Insertion of the transgene has led to a recessive-lethal phenotype, with no parent-of-origin effects on viability, suggesting that the transgene has not inserted into an imprinted region of the genome. HpaII and HhaI methylation sensitive restriction sites within the bacterial LacZ reporter gene are completely methylated when activity of the maternally inherited transgene is detected in the fetal liver, and not methylated when the paternally inherited transgene is silent. Thus DNA methylation of LacZ is correlated with maternal inheritance and may be implicated in the genomic imprinting mechanism as others have suggested. However, in contrast to the commonly found correlation of expression and low DNA methylation, the LacZ gene was expressed in fetal liver when fully methylated. This result may imply the existence of negative regulatory activities that recognize the unmethylated LacZ gene.

Heterodimerization among thyroid hormone receptor, retinoic acid receptor, retinoid X receptor, chicken ovalbumin upstream promoter transcription factor, and an endogenous liver protein.

Thyroid hormone receptor (TR) binds to DNA as a monomer, homodimer, and heterodimer with nuclear proteins. We have confirmed that the TR can heterodimerize with retinoid X receptors (RXRs)-alpha and -beta, and have found that another member of the nuclear receptor superfamily, chicken ovalbumin upstream promoter transcription factor (COUP-TF), also formed heterodimers with the TR in the context of binding to a palindromic thyroid hormone-responsive element (TREp). The interaction between COUP-TF and the TR was confirmed using specific antibodies which supershifted the COUP-TF/TR DNA complexes. The complex between the TR and the major TR heterodimerization partner in liver was unaffected by antibodies to COUP-TF and RXR beta, but was supershifted by an anti-RXR alpha antibody, indicating that the liver protein is highly related to RXR alpha. Indeed, the TR/RXR and TR/liver protein heterodimers contact the same guanidine residues in TREp. The retinoic acid receptor (RAR) also heterodimerized with COUP-TF as well as with RXR alpha, RXR beta, and the TR heterodimerization partner in liver. In contrast to its ability to heterodimerize with the TR and RAR, we did not detect heterodimers between COUP-TF and either RXR alpha, RXR beta, or the liver nuclear protein in the context of binding to the TREp. These results show that the major TR heterodimerization partner in liver is highly related to RXR alpha, but that other nuclear receptors such as COUP-TF can heterodimerize with the TR and RAR, suggesting that selective protein-protein interactions may be involved in the tissue and target gene specificities of hormone action.

Transgenic mice expressing a constitutively active retinoic acid receptor in the lens exhibit ocular defects.

Retinoic acid receptors (RARs) modulate gene expression following association with retinoic acid (RA). In transient transfection, an RAR alpha-beta-galactosidase fusion protein (RAR-LacZ) was able to transactivate expression in the absence of RA. When expressed in the ocular lens of transgenic mice, this constitutively active RAR-LacZ fusion gene resulted in founder and progeny animals that exhibited cataracts and microphthalmia, both being characteristics of retinoid-induced teratogenesis. The transgenic phenotypes indicate that retinoid teratogenesis can be mimicked by expression of a constitutively active RAR-LacZ fusion protein in retinoid-sensitive tissues.

Transgenic indicator mice for studying activated retinoic acid receptors during development.

Retinoic acid (RA) receptors (RARs) are ligand-inducible transcription factors that bind to specific DNA sequences associated with the regulatory regions of RA-regulatable genes. Since RA has been implicated as an important factor both in normal development and in teratological studies, one would like to have a model system that detects the presence of activated receptors during development. We have constructed a recombinant reporter gene that has three copies of the RA response element (RARE) from the RAR beta-2 promoter 5' to the herpes simplex virus thymidine kinase promoter; this regulatory region is coupled to the bacterial beta-galactosidase reporter gene. This construct was RA inducible in transient transfection assays in F9 embryonal carcinoma cells. Transgenic embryos with this reporter gene construct exhibited restricted and reproducible patterns of beta-galactosidase activity during embryogenesis, beginning between gestational ages day 7.5 and 8.5. At day 8.5, beta-galactosidase activity was detected in the closed neurotube and somites. Day 8.5 embryos, from pregnant females fed RA 14 hr earlier, exhibited a greater intensity and distribution of beta-galactosidase activity. Similarly, at later stages of gestation, maternal RA exposure resulted in enhanced embryonic beta-galactosidase expression. This type of transgenic indicator mouse should be useful in detailing the role of activated RARs during embryonic development.