Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Graded retinoid responses in the developing hindbrain.

The purpose of this study was to make an explicit test of the idea that a retinoid could act as a morphogen, differentially activating genes and specifying anteroposterior (a-p) level in the developing vertebrate central nervous system (CNS). Our approach was to characterize the concentration-dependent effects of retinoic acid (RA) on the neural expression of a set of a-p patterning genes, both in vivo and in an in vitro system for neural patterning. Our results indicate that a retinoid is unlikely to specify a-p level along the entire CNS. Instead, our data support the idea that the developing hindbrain may be patterned by a retinoid gradient. Sequentially more posterior hindbrain patterning genes were induced effectively by sequentially higher RA concentration windows. The most posterior CNS level induced under our RA treatment conditions corresponded to the most posterior part of the hindbrain.

Retinoid signalling and axial patterning during early vertebrate embryogenesis.

There are many indications that active retinoids are regulatory signals during vertebrate embryogenesis. Treating vertebrate embryos with retinoids can cause teratogenic defects, including specific derangements of the main body axis. Other data show that early vertebrate embryos contain physiologically relevant concentrations of active retinoids and express retinoid binding proteins and receptors; that knockouts of retinoid receptors can induce homeotic defects; and that relevant developmental control genes are regulated by retinoid response elements. Here, we discuss the possibility that retinoids are developmental signals which regulate axial patterning in the early vertebrate embryo.

Neural induction and patterning in embryos deficient in FGF signaling.

We have examined the role of FGFs in neural induction and posteriorization of the central nervous system (CNS). Using embryos micro-injected with dominant negative FGF receptor RNA (XFD), we show that a patterned CNS can still develop following inhibition of FGF signaling. The most severely affected embryos developed with strong posterior defects. In these embryos, head development and expression of a marker of forebrain and midbrain, and of markers of the hindbrain, occurred relatively normally. However, the expression levels of a posterior marker, Hoxb-9, were considerably reduced compared to those in control embryos. The results support the idea that FGFs are involved in inducing posterior development, but they suggest that other signals are also necessary for antero-posterior patterning of the primary body axis.

XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B.

COUP-TFs (Chicken Ovalbumin Upstream Promoter Transcription Factors) have been proposed to be negative regulators of retinoid receptor-mediated transcriptional activation. In a previous paper we reported the cloning of a Xenopus (x) COUP-TF (Matharu, P.J. and Sweeney, G.E. (1992) Biochim. Biophys. Acta 1129, 331-334). Here we describe the cloning of a second xCOUP-TF. Amino acid sequence comparison between these two Xenopus COUP-TFs revealed a high level of similarity. Extensive amino acid sequence conservation was found among all Drosophila, Xenopus, zebrafish and mammalian COUP-TF genes examined. Phylogenetic tree analyses indicate that the vertebrate COUP-TFs fall into three classes. The two Xenopus COUP-TF genes show similar temporal expression patterns: both are expressed from the end of gastrulation. In situ hybridization studies reveal complex expression patterns in the developing central nervous system (CNS), besides expression in the eye and in some mesodermal tissues. Retinoic acid (RA) treatment enhances xCOUP-TF-A expression in neurula stage embryos, whereas the expression of xCOUP-TF-B is inhibited during the same developmental period. The strictly conserved amino acid sequences and the strong similarities between the expression patterns of the two different xCOUP-TFs on the one hand, and other vertebrate COUP-TF homologues on the other, make it likely that COUP-TFs have a conserved role in patterning the nervous system.

Expression patterns of Hoxb genes in the Xenopus embryo suggest roles in anteroposterior specification of the hindbrain and in dorsoventral patterning of the mesoderm.

Hox genes are thought to participate in patterning the anteroposterior (a-p) axis during vertebrate embryogenesis. In this investigation, the spatial expression of six Hoxb genes was analyzed in early embryos of Xenopus laevis by in situ hybridization. Hoxb gene expression was first detected in late gastrulae/early neurulae, by which stage, the characteristic spatially colinear Hoxb gene expression sequence was already apparent. Dissection experiments indicated that the establishment of these localized expression patterns coincides with the acquisition of anteroposterior positional information along the main body axis. The Hoxb genes continued to be expressed in similar domains along the anteroposterior axis at all developmental stages examined, although there were some changes in expression at the cellular level. Interestingly, the 3' genes, Hoxb-1, Hoxb-3, and Hoxb-4 were expressed in very restricted domains in the future hindbrain, while Hoxb-5, Hoxb-7, and Hoxb-9 transcripts were present along the entire presumptive spinal cord. It was thus notable that the 5' Hoxb genes exhibited different types of expression domain than the 3' Hoxb genes. These observations suggest that there may be different mechanisms regulating the expression of the 3' and 5' Hoxb genes. Expression of all of the Hoxb genes analyzed, except Hoxb-4, was predominantly detectable in the central nervous system or in neural crest-derived structures. Hoxb-4 mRNA was detected in the central nervous system, but interestingly, the major expression site for this gene was the somites. The other Hoxb genes tested failed to show significant expression in the somitic mesoderm, although transcripts from genes 5' from Hoxb-4 were detected in other mesodermal tissues. In the vertebrate trunk, anteroposterior patterning of the CNS is thought to be regulated by the somites. The results obtained here for Xenopus embryos did not explicitly support the idea of a Hoxb code for the somites, although we cannot rule this out. Instead, interestingly, the data were consistent with a role for Hoxb genes in dorsoventral patterning of the mesoderm.

Overexpression of a cellular retinoic acid binding protein (xCRABP) causes anteroposterior defects in developing Xenopus embryos.

We have isolated the first Xenopus laevis cDNA coding for a cellular retinoic acid binding protein (xCRABP). xCRABP contains a single open reading frame, coding for an approximately 15 x 10(3) M(r) protein. Northern blot analysis shows that this cDNA hybridizes to a mRNA that is expressed both maternally and zygotically and which already reaches maximal expression during gastrulation (much earlier than previously described CRABP genes from other species). In situ hybridisation showed that at the onset of gastrulation, xCRABP mRNA is localised at the dorsal side of the embryo, in the ectoderm and in invaginating mesoderm. xCRABP expression then rapidly resolves into two domains; a neural domain, which becomes localised in the anterior hindbrain, and a posterior domain in neuroectoderm and mesoderm. These two domains were already evident by the mid-gastrula stage. We investigated the function of xCRABP by injecting fertilized eggs with an excess of sense xCRABP mRNA and examined the effects on development. We observed embryos with clear antero-posterior defects, many of which resembled the effects of treating Xenopus gastrulae with all-trans retinoic acid. Notably, the heart was deleted, anterior brain structures and the tail were reduced, and segmentation of the hindbrain was inhibited. The effects of injecting xCRABP transcripts are compatible with the idea that xCRABP overexpression modulates the action of an endogenous retinoid, thereby regulating the expression of retinoid target genes, such as Hox genes. In support of this, we showed that the expression of two Xenopus Hoxb genes, Hoxb-9 and Hoxb-4, is strongly enhanced by xCRABP over-expression. These results suggest that xCRABP expression may help to specify the anteroposterior axis during the early development of Xenopus laevis.

Xenopus blastulae show regional differences in competence for mesoderm induction: correlation with endogenous basic fibroblast growth factor levels.

Single cells from the animal cap and marginal zone (MZ) of mid-blastula stage embryos can undergo mesodermal or ectodermal differentiation as small clones under defined conditions in culture. Here we report that cells treated with Xenopus basic fibroblast growth factor (XbFGF), a mesoderm-inducing factor, usually differentiated into muscle. MZ cells, which normally give rise to most of the mesoderm, responded to lower concentrations of XbFGF than animal pole (AP) presumptive ectoderm cells. This difference in sensitivity correlated with immunocytochemical staining patterns that showed much greater levels of endogenous bFGF within MZ than AP cells in early embryos. At the mid-late blastula stage, nuclei of MZ cells were strongly immunoreactive. Nuclear staining persisted during gastrula and neurula stages, and extracellular bFGF also became apparent. Subsequently in somites, immunoreactivity of nuclei declined while that of the extracellular matrix was retained during tailbud stages. Nuclear localization of bFGF appeared to be temporally correlated with new transcription of muscle-specific genes, and extracellular bFGF was present during morphological differentiation. The results suggest that a cell's competence for mesoderm induction is related to its position in the embryo.

Nuclear translocation of fibroblast growth factor during Xenopus mesoderm induction.

Mesoderm induction, the earliest inductive cell-cell interaction in vertebrate embryogenesis, is thought to be mediated by polypeptide growth factors including fibroblast growth factor (FGF). Here we present an immunocytochemical analysis of FGF during mesoderm induction in Xenopus laevis. Antibodies to both basic and acidic FGF were immunoreactive with oocytes and early embryos. Immunostaining was predominantly intracellular and was concentrated in the marginal zone and vegetal pole throughout cleavage and blastula stages. In addition, basic FGF (bFGF) antibodies showed intense nuclear staining in these regions, at and following the mid-blastula transition, when embryonic transcription begins. Acidic FGF (aFGF) also appeared in some nuclei at these stages. Taken together the evidence suggests that FGF is prepositioned in mesoderm-forming regions and is actively involved in mesoderm induction in vivo.

Single cell analysis of mesoderm formation in the Xenopus embryo.

We have examined the developmental specification of individual cells in the Xenopus blastula using a new in vitro culture system. Regional differences are apparent at the mid-blastula stage when animal hemisphere cells form only ectodermal cell types, while many clones from below the pigment boundary contain mesodermal cell types. A number of clones give rise to more than one differentiated cell type indicating that the initial steps of mesoderm induction are potentially reversible. Animal hemisphere cells can be induced to form mesoderm by fibroblast growth factor (FGF). Different cell types predominate at different FGF concentrations and the neighbours in this sequence are also the pairs of cell types most usually associated in mixed clones derived from the marginal zone. We propose that the specification of individual cells depends upon both the concentration of inducing factor and on stochastic intracellular events.

A mesoderm-inducing factor produced by WEHI-3 murine myelomonocytic leukemia cells is activin A.

The first inductive interaction in amphibian development is mesoderm induction, during which a signal from the vegetal hemisphere of the blastula-staged embryo induces mesoderm from overlying equatorial cells. Recently, a number of 'mesoderm-inducing factors' (MIFs), which may be responsible for this interaction, have been discovered. Examples of these MIFs include members of the fibroblast growth factor family as well as members of the TGF-beta superfamily such as TGF-beta 2. In addition to these purified factors, several new sources of mesoderm-inducing activity have been described. One of the most potent of these is the murine myelomonocytic leukemia cell line WEHI-3. Even at high dilutions, conditioned medium from WEHI-3 cells induces isolated Xenopus animal pole regions to form a variety of mesodermal cell types. In this paper we show by several criteria, including N-terminal amino acid sequencing, Northern blotting and various functional assays, that the WEHI-MIF is activin A. Activins are known to modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and to cause the differentiation of two erythroleukemia cell lines. Our results, along with recent data from other laboratories, indicate that these molecules may also act in early development in the formation of the mesoderm.

Mesoderm induction by fibroblast growth factor in early Xenopus development.

In early amphibian development the mesoderm is formed around the equator of the blastula in response to inductive signals from the endoderm. At the time of its formation the mesoderm consists of a large 'ventral type' zone and a small 'organizer' zone. A screen of candidate substances showed that a small group of heparin binding growth factors (HBGFs) were active as mesoderm inducing agents in vitro. The fibroblast growth factors (aFGF and bFGF) and embryonal carcinoma derived growth factor (ECDGF) all show similar potency and can produce ventral inductions at concentrations above about 100 pm. Single blastula ectoderm cells can be induced and will differentiate in a defined medium to form mesodermal tissues and all inner blastula cells are competent to respond to the factors. Inducing activity can be extracted from Xenopus blastulae and can be purified by heparin affinity chromatography. Antibody neutralization and Western blotting experiments identify this activity as bFGF. The amounts present are small but would be sufficient to evoke ventral inductions in vivo. It is not yet known whether the bFGF is localized to the endoderm, although it is known that inducing activity secreted by endodermal cells can be neutralized by heparin. The competence of ectoderm to respond to FGF rises from about the 128-cell-stage and falls again by the onset of gastrulation. This change is paralleled by a rise and fall of binding of 125I-labelled aFGF. Chemical cross-linking reveals that this binding is attributable to a receptor of molecular mass about 130 kilodaltons (kDa). The receptor is present both in the marginal zone, which responds to the signal in vivo, and in the animal pole region, which is not induced in vivo but which will respond to HBGFs in vitro. In intact embryos we believe that the ventral type mesoderm forms the somites, kidney and other intermediate structures as well as the blood islands of the ventral midline. These intermediate structures are induced as a function of distance from the organizer in a process called 'dorsalization'. Lithium salts have a dorsalizing effect on whole embryos and also on explants from the ventral marginal zone, causing them to form large blocks of muscle. Lithium will also cause large muscle blocks to form when applied to ectoderm explants together with FGF. It is difficult to extend these results directly to mammalian embryos, but we have shown that the products of the murine int-2 gene and of the human k-fgf genes are active as mesoderm inducing factors.

Clonal analysis of mesoderm induction in Xenopus laevis.

Acidic fibroblast growth factor (aFGF) has been used to induce mesoderm from single animal pole cells of midblastula stage Xenopus embryos. The cells are individually cultured in a completely defined medium and are able to differentiate as small clones in a high proportion of cases. FGF-treated cells can give rise to several mesodermal cell types, while untreated cells show only epidermal or neural differentiation. Mesodermal differentiation can occur in clones of as few as eight cells, indicating that any additional cell-cell interactions required for mesodermal differentiation can be met by the medium used.

The role of fibroblast growth factor in early Xenopus development.

In early amphibian development, the mesoderm is formed around the equator of the blastula in response to an inductive signal from the endoderm. A screen of candidate substances showed that a small group of heparin-binding growth factors (HBGFs) were active as mesoderm-inducing agents in vitro. The factors aFGF, bFGF, kFGF and ECDGF all show similar potency and can produce inductions at concentrations above about 100 pM. The product of the murine int-2 gene is also active, but with a lower specific activity. Above the induction threshold there is a progressive increase of muscle formation with dose. Single blastula ectoderm cells can be induced and will differentiate in a defined medium to form mesodermal tissues. All inner blastula cells are competent to respond to the factors but outer cells, bearing oocyte-derived membrane, are not. Inducing activity can be extracted from Xenopus blastulae and binds to heparin like the previously described HBGFs. Antibody neutralization and Western blotting experiments identify this activity as bFGF. The amounts present are small but would be sufficient to evoke inductions in vivo. It is not yet known whether the bFGF is localized to the endoderm, although it is known that inducing activity secreted by endodermal cells can be neutralized by heparin. The competence of ectoderm to respond to HBGFs rises from about the 128-cell stage and falls again by the onset of gastrulation. This change is paralleled by a rise and fall of binding of 125I-aFGF. Chemical cross-linking reveals that this binding is attributable to a receptor of relative molecular mass about 130 x 10(3).(ABSTRACT TRUNCATED AT 250 WORDS)

Mesoderm-inducing factors: a small class of molecules.

Mesoderm-inducing factors (MIF's) from chick embryos, XTC cells and WEHI-3 cells were studied using various procedures. The object was to find whether they are similar to heparin-binding growth factors (HBGFs-the only known pure mesoderm-inducing substances) and, if not, whether they are similar to each other. The major active components from all three MIF sources behave as somewhat hydrophobic, acid-stable molecules and do not bind to heparin. They all have relative molecular masses of about 13,000 measured by HPLC size exclusion chromatography. The isoelectric points measured by chromatofocusing were 6.7 (WEHI) and 7.7 (XTC). The chick MIF seemed somewhat heterogeneous by chromatofocusing and a portion of its activity bound to heparin sepharose. All three MIFs have similar effects on explants of Xenopus blastula ectoderm to the heparin-binding growth factors, causing an elongation at the time of gastrulation followed by the development of mesenchyme, mesothelium and muscle cells, the proportion of muscle increasing with dose. Unlike the HBGFs they all also induce notochord if sufficiently high concentrations are used. Our study shows that the MIFs examined here form a small group of potent agents distinct from the HBGFs and from other known growth and differentiations factors. Their occurrence in various tissues and cell lines suggests that they have functions in the adult organism as well as during early development.

Mesoderm induction in early Xenopus embryos by heparin-binding growth factors.

In early embryonic development the basic body plan arises because cells in different regions become programmed to follow different developmental pathways. We have proposed that in the early amphibian embryo this process of regional specification arises from the action of three different inducing factors, or morphogens, but we have not until now had any idea of their chemical nature. In this paper we report that pure basic fibroblast growth factor (bFGF), at very low concentrations and with high specificity, closely mimics the effect of the ventrovegetal (VV) signal and that the transmission of the natural VV signal can be blocked by heparin, suggesting that it may be a heparin-binding factor such as bFGF.

The appearance and distribution of intermediate filament proteins during differentiation of the central nervous system, skin and notochord of Xenopus laevis.

Antibodies against various intermediate filament proteins have been used to follow cell differentiation in the early Xenopus embryo. Three new monoclonal antibodies against Xenopus cytokeratins raised against Triton-insoluble material from tadpoles (RD35/2a, RD35/3a and D3/3a), two antibodies against mammalian cytokeratins (LE65 and LP3K), monoclonal anti-(rat 200 K neurofilament protein), rabbit anti-(rat glial filament acidic protein), and rabbit antibodies to hamster and calf vimentin were used. We show that cytokeratins are present in the early central nervous system (CNS) and persist in the ependymal cells of the adult CNS. We also show that the notochord contains cytokeratin. The ontogeny of intermediate filament protein appearance in the CNS, skin and notochord between neural fold stage and swimming tadpole stage are described. These results are discussed in particular with regard to the use of the antibodies as differentiation markers.