The ADHD-susceptibility gene lphn3.1 modulates dopaminergic neuron formation and locomotor activity during zebrafish development.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, increased impulsivity and emotion dysregulation. Linkage analysis followed by fine-mapping identified variation in the gene coding for Latrophilin 3 (LPHN3), a putative adhesion-G protein-coupled receptor, as a risk factor for ADHD. In order to validate the link between LPHN3 and ADHD, and to understand the function of LPHN3 in the etiology of the disease, we examined its ortholog lphn3.1 during zebrafish development. Loss of lphn3.1 function causes a reduction and misplacement of dopamine-positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. The behavioral phenotype can be rescued by the ADHD treatment drugs methylphenidate and atomoxetine. Together, our results implicate decreased Lphn3 activity in eliciting ADHD-like behavior, and demonstrate its correlated contribution to the development of the brain dopaminergic circuitry.

Cloning and developmental expression of zebrafish GTP cyclohydrolase I.

GTP cyclohydrolase I (GCH) catalyses the conversion of GTP to dihydroneopterin triphosphate, initiating the pteridine pathway. The final product tetrahydrobiopterin (H4biopterin) is the cofactor for neurotransmitter synthesis and for tyrosine supply during melanogenesis. Sepiapterin accumulates as a pigment. We cloned the zebrafish gch cDNA, which encodes a protein highly homologous to other vertebrate sequences and characterised the recombinant enzyme. By in situ hybridisation, we found that gch is expressed in both the melanophore and xanthophore lineages during early development. gch-expression almost disappears after 3 days post-fertilisation (dpf), despite further sepiapterin synthesis. gch-transcripts are also located in catecholaminergic neurons, within the central nervous system and in the arch-associated neurons.

The zebrafish buttonhead-like factor Bts1 is an early regulator of pax2.1 expression during mid-hindbrain development.

Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, we have searched for vertebrate genes related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. We report here the identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) and shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrate that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. Our results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor.

Zebrafish lunatic fringe demarcates segmental boundaries.

Cell interactions involving Notch signaling are required for the demarcation of tissue boundaries in both invertebrate and vertebrate development. Members of the Fringe gene family encode beta-1,3 N-acetyl-glucosaminyltransferases that function to refine the spatial localization of Notch-receptor signaling to tissue boundaries. In this paper we describe the isolation and characterization of the zebrafish (Danio rerio) homologue of the lunatic fringe gene (lfng). Zebrafish lfng is generally expressed in equivalent structures to those reported for the homologous chick and mouse genes. These sites include expression along the A-P axis of the neural tube, within the lateral plate mesoderm, in the presomitic mesoderm and the somites and in specific rhombomeres of the hindbrain; however, within these general expression domains species-specific differences in lfng expression exist. In mouse, Lfng is expressed in odd-numbered rhombomeres, whereas in zebrafish, expression occurs in even-numbered rhombomeres. In contrast to reports in both mouse and chicken embryos showing a kinematic cyclical expression of Lfng mRNA in the presomitic paraxial mesoderm, we find no evidence for a cyclic pattern of expression for the zebrafish lfng gene; instead, the zebrafish lfng is expressed in two static stripes within the presomitic mesoderm. Nevertheless, in zebrafish mutants affecting the correct formation of segment boundaries in the hindbrain and somites, lfng expression is aberrant or lost.

Neural plate patterning: upstream and downstream of the isthmic organizer.

Two organizing centres operate at long-range distances within the anterior neural plate to pattern the forebrain, midbrain and hindbrain. Important progress has been made in understanding the formation and function of one of these organizing centres, the isthmic organizer, which controls the development of the midbrain and anterior hindbrain. Here we review our current knowledge on the identity, localization and maintenance of the isthmic organizer, as well as on the molecular cascades that underlie the activity of this organizing centre.

Coregulation of anterior and posterior mesendodermal development by a hairy-related transcriptional repressor.

During embryonic development in vertebrates, the endoderm becomes patterned along the anteroposterior axis to produce distinct derivatives. How this regulation is controlled is not well understood. We report that the zebrafish hairy/enhancer of split [E(spl)]-related gene her5 plays a critical role in this process. At gastrulation, following endoderm induction and further cell interaction processes including a local release of Notch/Delta signaling, her5 expression is progressively excluded from the presumptive anterior- and posteriormost mesendodermal territories to become restricted to an adjacent subpopulation of dorsal endodermal precursors. Ectopic misexpressions of wild-type and mutant forms of her5 reveal that her5 functions primarily within the endodermal/endmost mesendodermal germ layer to inhibit cell participation to the endmost-fated mesendoderm. In this process, her5 acts as an active transcriptional repressor. These features are strikingly reminiscent of the function of Drosophila Hairy/E(spl) factors in cell fate decisions. Our results provide the first model for vertebrate endoderm patterning where an early regulatory step at gastrulation, mediated by her5 controls cell contribution jointly to the anterior- and posteriormost mesendodermal regions.

Cloning of zebrafish activin type IIB receptor (ActRIIB) cDNA and mRNA expression of ActRIIB in embryos and adult tissues.

A full-length cDNA encoding for activin type IIB receptor (ActRIIB) was cloned from zebrafish embryos. It encodes a protein with 509 amino acids consisting of a signal peptide, an extracellular ligand binding domain, a single transmembrane region, and an intracellular kinase domain with predicted serine/threonine specificity. The extracellular domain shows 74-91% sequence identity to human, bovine, mouse, rat, chicken, Xenopus and goldfish activin type IIB receptors, while the transmembrane region and the kinase domain show 67-78% and 82-88% identity to these known activin IIB receptors, respectively. In adult zebrafish, ActRIIB mRNA was detected by RT-PCR in the gonads, as well as in non-reproductive tissues, including the brain, heart and muscle. In situ hybridization on ovarian sections further localized ActRIIB mRNA to cytoplasm of oocytes at different stages of development. Using whole-mount in situ hybridization, ActRIIB mRNA was found to be expressed at all stages of embryogenesis examined, including the sphere, shield, tail bud, and 6-7 somite. These results provide the first evidence that ActRIIB mRNA is widely distributed in fish embryonic and adult tissues. Cloning of zebrafish ActRIIB demonstrates that this receptor is highly conserved during vertebrate evolution and provides a basis for further studies on the role of activin in reproduction and development in lower vertebrates.

Characterization of the zebrafish Orb/CPEB-related RNA binding protein and localization of maternal components in the zebrafish oocyte.

The animal/vegetal axis of the zebrafish egg is established during oogenesis, but the molecular factors responsible for its specification are unknown. As a first step towards the identification of such factors, we present here the first demonstration of asymmetrically distributed maternal mRNAs in the zebrafish oocyte. To date, we have distinguished three classes of mRNAs, characterized by the stage of oocyte maturation at which they concentrate to the future animal pole. We have further characterized one of these mRNAs, zorba, which encodes a homologue of the Drosophila Orb and Xenopus CPEB RNA-binding proteins. Zorba belongs to the group of earliest mRNAs to localize at the animal pole, where it becomes restricted to a tight subcortical crescent at stage III of oogenesis. We show that this localization is independent of microtubules and microfilaments, and that the distribution of Zorba protein parallels that of its mRNA.

Molecular cloning of Zcoe2, the zebrafish homolog of Xenopus Xcoe2 and mouse EBF-2, and its expression during primary neurogenesis.

Xcoe2 is a recently identified HLH transcription factor of the Xenopus primary neurogenesis pathway, which is necessary downstream of Neurogenin to stabilize neuroblast determination (Dubois, L. et al., 1998. Curr. Biol. 8, 199-209). We report here the embryonic expression pattern of Zcoe2, its zebrafish homolog. As observed for Xcoe2, Zcoe2 is strongly expressed in a subset of the neurogenin1- (ngn1-) positive primary neuroblasts of the spinal cord. In the anterior neural plate, in contrast, Zcoe2 is expressed earlier and more widely than ngn1. This pattern is strongly maintained in the presumptive mesencephalon and rhombomeres 1-4 until the 2-3-somite stage. This expression of Zcoe2 in the brain anlage calls for a re-analysis in zebrafish of the functional relationship demonstrated in Xenopus between Coe2 and Neurogenin factors. At later stages, Zcoe2 is expressed in early forming neurons of the anterior brain and is a marker of the olfactory placodes.

XCoe2, a transcription factor of the Col/Olf-1/EBF family involved in the specification of primary neurons in Xenopus.

BACKGROUND: Primary neurogenesis in Xenopus is a model for studying the control of neural cell fate decisions. The specification of primary neurons appears to be driven by transcription factors containing a basic region and a helix-loop-helix (HLH) motif: expression of Xenopus neurogenin-related-1 (X-ngnr-1) defines the three prospective domains of primary neurogenesis, and expression of XNeuroD coincides with neuronal differentiation. The transition between neuronal competence and stable commitment to a neuronal fate remains poorly characterised, however. RESULTS: Drosophila Collier and rodent early B-cell factor/olfactory-1 define a family of HLH transcription factors containing a previously unknown type of DNA-binding domain. We isolated an orthologous gene from Xenopus, Xcoe2, which is expressed in precursors of primary neurons. Xcoe2 is transcribed after X-ngnr-1 and before XNeuroD. Overexpression of a dominant-negative mutant of XCoe2 prevented neuronal differentiation. Conversely, overexpressed wild-type Xcoe2 could promote ectopic differentiation of neurons, in both the neural plate and the epidermis. In contrast to studies with X-ngnr-1 or XNeuroD, the supernumerary neurons induced by Xcoe2 appeared in a 'salt-and-pepper' pattern, resulting from the activation of X-Delta1 expression and feedback regulation by lateral inhibition. CONCLUSIONS: XCoe2 may play a pivotal role in the transcriptional cascade that specifies primary neurons in Xenopus embryos: by maintaining Delta-Notch signalling, XCoe2 stabilises the higher neural potential of selected progenitor cells that express X-ngnr-1, ensuring the transition between neural competence and irreversible commitment to a neural fate; and it promotes neuronal differentiation by activating XNeuroD expression, directly or indirectly.

Transcription factors and head formation in vertebrates.

Evidence from Drosophila and also vertebrates predicts that two different sets of instructions may determine the development of the rostral and caudal parts of the body. This implies different cellular and inductive processes during gastrulation, whose genetic requirements remain to be understood. To date, four genes encoding transcription factors expressed in the presumptive vertebrate head during gastrulation have been studied at the functional level: Lim-1, Otx-2, HNF-3 beta and goosecoid. We discuss here the potential functions of these genes in the formation of rostral head as compared to posterior head and trunk, and in the light of recent fate map and expression analyses in mouse, chick, Xenopus and zebrafish. These data indicate that Lim-1, Otx-2 and HNF-3 beta may be involved in the same genetic pathway controlling the formation of the prechordal mesendoderm, which is subsequently required for rostral head development. goosecoid may act in a parallel pathway, possibly in conjunction with other, yet unidentified, factors.

Involvement of Wnt-1 in the formation of the mes/metencephalic boundary.

Wnt-1, a putative signaling molecule, is required before the 7 somite stage (E8.5) for the development of midbrain structures in the mouse. We show here that Wnt-1 is also needed for the formation of a boundary between the mesencephalic and metencephalic domains of the neural tube. In embryos homozygous for the Wnt-1sw allele, mesencephalic and metencephalic markers fail to segregate and the establishment of a straight limit of Otx-2 and Wnt-1 expression at the mid-hindbrain junction is impaired. In addition, as observed previously in heterotopic mes/metencephalic transplantation experiments in avian embryos, Wnt-1 expression is induced at the border of ectopic mes- and metencephalic islands observed in Wnt-1sw/sw mutants, suggesting that, in situ, interactions between mes- and metencephalic cells reinforce Wnt-1 expression at the boundary.

Determination events in the nervous system of the vertebrate embryo.

Molecular and functional data suggest that the regionalization of the caudal portion of the vertebrate embryonic brain (hindbrain) is set up through a process of segmentation. In contrast, the more rostrally located met-mesencephalic domain (mid-hindbrain junction) appears to follow a mode of specification that relies on long-range inducing and organizing activities originating from the central region of the domain. Recent studies addressing this mode of anterior/posterior determination point to Wnt-1 as a key player in this process.

c-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo.

We cloned the chick homologue of the mouse Otx2 gene, c-otx2, and analyzed its expression pattern during gastrulation. During mouse embryogenesis, Otx2 expression is first detected in the entire epiblast and after the formation of the primitive streak becomes confined to the most anterior region of the embryo corresponding to presumptive fore- and mid-brain. Similarly, two distinct phases of c-otx2 expression were observed in the chick. c-otx2 transcripts were first detected in the unincubated egg and up to stage XIII, in all epiblast, and forming hypoblast and mesoblast cells. During primitive streak progression, c-otx2 expression becomes progressively restricted to anterior regions and is mainly associated with Hensen's node. When the extension of the streak is maximal, transcripts are only found in Hensen's node. A second phase of c-otx2 expression starts during streak regression. c-otx2 transcripts are lost from the node and present in higher abundance in anterior neuroectoderm and mesendoderm, with the exception of forming notochord and floor plate. The first phase of expression bears strong similarity with that of c-gsc, a gene shown to be a marker for cells that have organizer activity in the chick. Therefore, we compared the expression of the two genes by double staining on the same embryo. This analysis demonstrated that c-otx2 is transcribed first and its expression in the hypoblast precedes that of c-gsc. On the other hand, c-gsc is an earlier marker of primitive streak cells. The expression domains of the two genes transiently overlap in Hensen's node and anterior mesendoderm, whereas only c-otx2 is expressed in neuroectodermal areas. The second phase of c-otx2 expression is sensitive to an early treatment with retinoic acid. This treatment abolishes c-otx2 expression in mesendoderm and restricts it to most anterior regions in the forming neural plate. In conclusion, our results suggest that c-otx2 expression is first associated with cells with an anterior mesendoderm fate and subsequently extends to anterior neuroectoderm.

Ectopic induction and reorganization of Wnt-1 expression in quail/chick chimeras.

When grafted ectopically into the diencephalon of a chick host embryo, a portion of met-mesencephalon straddling the met-mesencephalic constriction has the capacity to induce En-2 expression in the surrounding host tissue. Subsequently, tectal and cerebellar structures, composed of both host and grafted cells, are reconstructed in this ectopic location at the expense of the host diencephalon. Previous experiments indicated that the induction of En-2 was correlated with Wnt-1 expression within the graft. The aim of the present study was: (i) to determine whether Wnt-1 expression was spatially regulated within the graft, (ii) to investigate whether host Wnt-1-expressing cells were also involved in the ectopic met-mesencephalic development and, if so, (iii) to localize these Wnt-1-positive domains in relation to the patterning of the ectopically developing met-mesencephalic territory. We studied the expression profile of Wnt-1, in relation with that of other positional markers, in quail/chick chimeras where various portions of met-mesencephalon had been grafted into the diencephalon. We found that Wnt-1 expression was reorganized within the graft, and that it was also induced in the host in contact with the graft. Moreover, these ectopic expressions of Wnt-1, in both the grafted and the surrounding host tissues, were organized in concert to form a continuous positive line at the host/graft junction, the location of which depended on the precise origin of the graft. Finally, we found that this line was frequently located at the limit between territories expressing different positional markers. We propose that Wnt-1 expression is turned on at the junction between domains of different phenotypes, and may be used as a border to stabilize these adjacent differently committed territories.

A short 5'-flanking region mediates glucose repression of amylase gene expression in Drosophila melanogaster.

Expression of the alpha-amylase gene is highly repressed by dietary glucose in Drosophila melanogaster larvae. Here, we show that glucose repression is controlled by DNA sequences that are located upstream of the transcribed region. Recombinant gene constructions, in which the amylase promoter sequences were fused with the transcribed region of the Adh gene, were expressed in transgenic Drosophila larvae. The expression of ADH from the recombinant gene was shown to be subject to glucose repression. The function of potential regulatory cis-acting elements within the glucose responsive upstream region was examined by deletion analysis and by site-directed mutagenesis, coupled with expression assays in transformed larvae. The upstream deletion analysis showed that essential elements, both for overall activity and for glucose repression of the amylase gene, are located within a 109-bp region upstream of the transcription start site. Site-directed mutagenesis of these upstream sequences showed that the TATA motif, at position -31, and a novel 36-bp element, at position -109, were necessary for full activity of the amylase promoter. None of the introduced mutations resulted in loss of glucose responsiveness. These results indicate that glucose repression, in Drosophila, is mediated by transcriptional mechanisms that involve multiple, functionally redundant DNA elements.

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster.

Previous studies have demonstrated that the expression of the alpha-amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the alpha-amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.

The mouse NCAM gene displays a biphasic expression pattern during neural tube development.

The neural cell adhesion molecule (NCAM) is one of the most abundant cell adhesion molecules expressed in vertebrates and it is thought to play important roles as a regulator of morphogenetic processes, but little is known of its expression pattern in mammalian embryos. In this study, we have examined the developmental profile of NCAM gene expression in mouse embryos from gestational day 7.5 to 12.5, focusing on the developing neural tube. NCAM transcripts were first detected around day 8.5 in the somites and the forming neural tube. At this stage, NCAM transcripts were expressed in the neuroepithelium throughout the width of the neural groove and tube up to a rostral boundary within the hindbrain, whereas NCAM mRNA levels were very low or undetectable in the neuroepithelium of the head region. The positional restriction of NCAM expression was confirmed by immunohistochemistry at the protein, and by polymerase chain reaction analysis at the RNA level. Expression in the neuroepithelium was transient as the level of NCAM transcripts declined in the germinal layer beyond day 8.5. By day 9.5, strong NCAM expression had appeared on the earliest postmitotic neurones along the entire neuraxis, and this pattern of expression in all regions with differentiating neurones was maintained until day 12.5. We conclude that NCAM expression in the neural tube occurs in two spatiotemporal distinct waves: a first wave in the proliferating neuroepithelium showing positional dependence along the rostrocaudal axis, and a second wave on essentially all neurones that have become postmitotic.

Regional specification during cerebellar development.

At early phases of development (stage HH12 of chick and quail embryos) the rostral part of the cerebellar anlage extends into the caudal third of the mesencephalic vesicle. This region is characterized by its high level of expression of the Wnt-1 and engrailed genes, which encode, respectively, for a secreted molecule and for a homeodomain protein. In order to analyze the potentialities of various parts of the cerebellar anlage, pieces of quail or mouse embryonic neural tubes have been transplanted heterotopically in the prosencephalon of HH12 chick embryos. These experiments indicated that signals operating in the plane of the neural tube are still operating in the donor avian and murine embryos. The met- mes- and prosencephalic territories of HH12 chick embryos are still competent to change their fates under the influence of these signals. It was also found that the Wnt-1 expression domain, the caudal mesencephalic vesicle, has striking organizing properties until late stages of development in avian and rodent embryos. The exact role of Wnt-1 in these inductive interactions is still unknown. Wnt-1 influence on cell fate could be mediated by the engrailed genes. Observations suggesting a later role of en-2 in the regionalization of the cerebellar cortex are also reported.