An inherited functional circadian clock in zebrafish embryos.

Circadian clocks are time-keeping systems found in most organisms. In zebrafish, expression of the clock gene Period3 (Per3) oscillates throughout embryogenesis in the central nervous system and the retina. Per3 rhythmic expression was free-running and was reset by light but not by the developmental delays caused by low temperature. The time of fertilization had no effect on Per3 expression. Per3 messenger RNA accumulates rhythmically in oocytes and persists in embryos. Our results establish that the circadian clock functions during early embryogenesis in zebrafish. Inheritance of maternal clock gene products suggests a mechanism of phase inheritance through ovogenesis.

D4 Dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour.

Zebrafish, a model developmental genetic organism, is being increasingly used in behavioural studies. We have initiated studies designed to evaluate the response of zebrafish to antipsychotic drugs. This study focuses on characterization of zebrafish D4 dopamine receptors (D4Rs) and the response of larval zebrafish to the atypical antipsychotic clozapine. The D4R is of interest because of its high affinity for clozapine, while interest in clozapine stems from its effectiveness in reducing symptoms in acutely psychotic, treatment-resistant schizophrenic patients. By mining the zebrafish genomic database, we identified three distinct D4R genes, drd4a, drd4b and drd4c, and generated full-length open reading frames encoding each of the three D4Rs by reverse transcription-polymerase chain reaction. Gene mapping studies showed that each D4R gene mapped to a distinct chromosomal location in the zebrafish genome, and each gene exhibited a unique expression profile during embryogenesis. When administered to larval zebrafish, clozapine produced a rapid and profound effect on locomotor activity. The effect of clozapine was dose-dependent, resulted in hypoactivity and was prevented by the D4-selective agonist ABT-724. Our data suggest that the inhibitory effect of clozapine on the locomotor activity of larval zebrafish may be mediated through D4Rs.

Sequence and expression pattern of ziro7, a novel, divergent zebrafish iroquois homeobox gene.

We have isolated the zebrafish ziro7 gene, a novel, divergent member of the Iroquois family. ziro7 is expressed at early epiboly stages in the dorsal half of the zebrafish embryo, with a higher level in the dorso-lateral margin. From mid-gastrulation stages onward, ziro7 is expressed in a large transversal stripe in the future neural plate, which subsequently divides into thinner stripes located in the diencephalon, midbrain and hindbrain.

Pattern formation in the lateral line of zebrafish.

The lateral line of fish and amphibians is a sensory system that comprises a number of individual sense organs, the neuromasts, arranged in a defined pattern on the surface of the body. A conspicuous part of the system is a line of organs that extends along each flank (and which gave the system its name). At the end of zebrafish embryogenesis, this line comprises 7-8 neuromasts regularly spaced between the ear and the tip of the tail. The neuromasts are deposited by a migrating primordium that originates from the otic region. Here, we follow the development of this pattern and show that heterogeneities within the migrating primordium prefigure neuromast formation.

Zebrafish evx1 is dynamically expressed during embryogenesis in subsets of interneurones, posterior gut and urogenital system.

The even-skipped-related homeobox genes (evx) are widely distributed through animal kingdom and are thought to play key role in posterior body patterning and neurogenesis. We have cloned and analyzed the expression of evx1 in zebrafish (see also Borday et al. (Dev. Dyn. 220 (2001) in press) which displays a dynamic and restricted expression pattern during neurogenesis. In spinal cord, rhombencephalon, and epiphysis, evx1 is expressed in several subsets of emerging interneurones prior to their axonal outgrowth, identified as primary interneurones and a subset of Pax2.1(+) commissural interneurones. In the hindbrain, evx1 is expressed in reticulospinal interneurones of rhombomeres 5 and 6 as well as in rhombomere 7 interneurones. The latest emerging evx1(+) interneurones in the hindbrain correspond to commissural interneurones. evx1 is also dynamically transcribed during the formation of the posterior gut and the uro-genital system in mesenchymal cells that border the pronephric ducts, the wall of the pronephric duct, and later in the posterior gut and the wall of the uro-genital opening. In larvae, the ano-rectal epithelium and the muscular layer that surrounds the analia-genitalia region remain stained up to 27 days. In contrast other vertebrates, evx1displays no early nor caudal expression in zebrafish.

Asymmetric expression of antivin/lefty1 in the early chick embryo.

Mammalian lefty and zebrafish antivin, highly related to lefty, are shown to be expressed asymmetrically and involved in the specification of the left body side of early embryos. We isolated a chick homologue of the antivin/lefty1 cDNA and studied its expression pattern during early chick development. We found that antivin/lefty1 is expressed asymmetrically on the left side of the prospective floorplate, notochord and lateral plate mesoderm of the chick embryo.

Calsenilin is required for endocrine pancreas development in zebrafish.

Calsenilin/DREAM/Kchip3 is a neuronal calcium-binding protein. It is a multifunctional protein, mainly expressed in neural tissues and implicated in regulation of presenilin processing, repression of transcription, and modulation of A-type potassium channels. Here, we performed a search for new genes expressed during pancreatic development and have studied the spatiotemporal expression pattern and possible role of calsenilin in pancreatic development in zebrafish. We detected calsenilin transcripts in the pancreas from 21 somites to 39 hours postfertilization stages. Using double in situ hybridization, we found that the calsenilin gene was expressed in pancreatic endocrine cells. Loss-of-function experiments with anti-calsenilin morpholinos demonstrated that injected morphants have a significant decrease in the number of pancreatic endocrine cells. Furthermore, the knockdown of calsenilin leads to perturbation in islet morphogenesis, suggesting that calsenilin is required for early islet cell migration. Taken together, our results show that zebrafish calsenilin is involved in endocrine cell differentiation and morphogenesis within the pancreas.

Dorsoventral development of the Drosophila embryo is controlled by a cascade of transcriptional regulators.

Maternal genes involved in dorsoventral (D/V) patterning of the Drosophila embryo interact to establish a stable nuclear concentration gradient of the Dorsal protein which acts as the morphogen along this axis. This protein belongs to the rel proto-oncogene and NF-KB transcriptional factor family and acts by controlling zygotic gene expression. In the ventral part of the embryo, dorsal specifically activates transcription of the gene twist and ventrally and laterally dorsal represses the expression of zerknüllt, a gene involved in the formation of dorsal derivatives. The extent of dorsal action is closely related to the affinity and the number of dorsal response elements present in these zygotic gene promoters. twist is one of the first zygotic genes necessary for mesoderm formation. It codes for a 'b-HLH' DNA-binding protein which can dimerize and bind to DNA in vitro and to polytene chromosomes in vivo. In addition, in cultured cells twist has been shown to be a transcriptional activator. Thus, the first events of embryonic development along the D/V axis are controlled at the transcriptional level.

Antivin, a novel and divergent member of the TGFbeta superfamily, negatively regulates mesoderm induction.

Mesoderm induction and patterning are mediated by members of the TGFbeta superfamily. We have isolated a novel zebrafish member, antivin, that structurally is highly related to mouse lefty. Overexpression of antivin completely abolishes mesoderm induction at blastula stage, yet resultant embryos develop well-patterned epidermal and neural derivatives. The mesoderm-inhibiting activity of antivin can be mimicked by lefty and is suppressed by increasing levels of the mesodermal inducer Activin or its receptors. On the basis of its expression and activity, we propose that Antivin normally functions as a competitive inhibitor of Activin to limit mesoderm induction in the early embryo.

Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process.

The origin of resident (noninflammatory) macrophages in vertebrate tissues is still poorly understood. In the zebrafish embryo, we recently described a specific lineage of early macrophages that differentiate in the yolk sac before the onset of blood circulation. We now show that these early macrophages spread in the whole cephalic mesenchyme, and from there invade epithelial tissues: epidermis, retina, and brain--especially the optic tectum. In the panther mutant, which lacks a functional fms (M-CSF receptor) gene, early macrophages differentiate and behave apparently normally in the yolk sac, but then fail to invade embryonic tissues. Our video recordings then document for the first time the behavior of macrophages in the invaded tissues, revealing the striking propensity of early macrophages in epidermis and brain to wander restlessly among epithelial cells. This unexpected behavior suggests that tissue macrophages may be constantly "patrolling" for immune and possibly also developmental and trophic surveillance. At 60 h post-fertilization, all macrophages in the brain and retina undergo a specific phenotypic transformation, into "early (amoeboid) microglia": they become more highly endocytic, they down-regulate the L-plastin gene, and abruptly start expressing high levels of apolipoprotein E, a well-known neurotrophic lipid carrier.

Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo.

The dorsoventral polarity of the vertebrate embryo is established through interactions between ventrally expressed bone morphogenetic proteins and their organizer-borne antagonists Noggin, Chordin, and Follistatin. While the opposing interactions between Short Gastrulation/Chordin and Decapentaplegic/BMP4 have been evolutionarily conserved in arthropods and vertebrates, there has been up to now no functional evidence of an implication of Noggin in the early patterning of organisms other than Xenopus. We have studied the contribution of Noggin to the embryonic development of the zebrafish. While single-copy noggin genes have been characterized in several vertebrate species, we report that the zebrafish genome harbors three noggin homologues. Overexpression experiments show that Noggin1, Noggin2, and Noggin3 can antagonize ventralizing BMPs. While all three factors have similar biological activities, their embryonic expression is different. The combined expression of the three genes recapitulates the different aspects of the expression of the single-copy noggin genes of other organisms. This suggests that the three zebrafish noggin genes and the single noggin genes of other vertebrates have evolved from a common ancestor and that subsequent differential loss of tissue-specific elements in the promoters of the different zebrafish genes accounts for their more restricted spatiotemporal expression. Finally we show that noggin1 is expressed in the fish organizer and able to dorsalize the embryo, suggesting its implication in the dorsoventral patterning of the zebrafish.

Ontogeny and behaviour of early macrophages in the zebrafish embryo.

In the zebrafish embryo, the only known site of hemopoieisis is an intra-embryonic blood island at the junction between trunk and tail that gives rise to erythroid cells. Using video-enhanced differential interference contrast microscopy, as well as in-situ hybridization for the expression of two new hemopoietic marker genes, draculin and leucocyte-specific plastin, we show that macrophages appear in the embryo at least as early as erythroid cells, but originate from ventro-lateral mesoderm situated at the other end of the embryo, just anterior to the cardiac field. These macrophage precursors migrate to the yolksac, and differentiate. From the yolksac, many invade the mesenchyme of the head, while others join the blood circulation. Apart from phagocytosing apoptotic corpses, these macrophages were observed to engulf and destroy large amounts of bacteria injected intravenously; the macrophages also sensed the presence of bacteria injected into body cavities that are isolated from the blood, migrated into these cavities and eradicated the microorganisms. Moreover, we observed that although only a fraction of the macrophage population goes to the site of infection, the entire population acquires an activated behaviour, similar to that of activated macrophages in mammals. Our results support the notion that in vertebrate embryos, macrophages endowed with proliferative capacity arise early from the hemopoietic lineage through a non-classical, rapid differentiation pathway, which bypasses the monocytic series that is well-documented in adult hemopoietic organs.

A role for FGF-8 in the dorsoventral patterning of the zebrafish gastrula.

Signals released from Spemann's organizer, together with ventralizing factors such as BMPs, are necessary to pattern the dorsoventral axis of the vertebrate embryo. We report that a member of the FGF family, fgf-8, not secreted by the axial mesoderm but expressed in a dorsoventral gradient at the margin of the zebrafish gastrula, also contributes to the establishment of the dorsoventral axis of the embryo. Ectopic expression of FGF-8 leads to the expansion of dorsolateral derivatives at the expense of ventral and posterior domains. Moreover, FGF-8 displays some organizer properties as it induces the formation of a partial secondary axis in the absence of factors released from Spemann's organizer territory. Analysis of its interaction with the ventralizing factors, BMPs, reveals that overexpression of FGF-8 inhibits the expression of these factors in the ventral part of the embryo as early as blastula stage, suggesting that FGF-8 acts upstream of BMP2 and BMP4. We conclude that FGF-8 is involved in defining dorsoventral identity and is an important organizing factor responsible for specification of mesodermal and ectodermal dorsolateral territories of the zebrafish gastrula.

Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo.

Definition of cell fates along the dorso-ventral axis depends on an antagonistic relationship between ventralizing transforming growth factor-beta superfamily members, the bone morphogenetic proteins and factors secreted from the dorsal organizer, such as Noggin and Chordin. The extracellular binding of the last group to the bone morphogenetic proteins prevents them from activating their receptors, and the relative ventralizer:antagonist ratio is thought to specify different dorso-ventral cell fates. Here, by taking advantage of a non-genetic interference method using a specific competitive inhibitor, the Lefty-related gene product Antivin, we provide evidence that cell fate along the antero-posterior axis of the zebrafish embryo is controlled by the morphogenetic activity of another transforming growth factor-beta superfamily subgroup--the Activin and Nodal-related factors. Increasing antivin doses progressively deleted posterior fates within the ectoderm, eventually resulting in the removal of all fates except forebrain and eyes. In contrast, overexpression of activin or nodal-related factors converted ectoderm that was fated to be forebrain into more posterior ectodermal or mesendodermal fates. We propose that modulation of intercellular signalling by Antivin/Activin and Nodal-related factors provides a mechanism for the graded establishment of cell fates along the antero-posterior axis of the zebrafish embryo.

A zebrafish nanos-related gene is essential for the development of primordial germ cells.

Asymmetrically distributed cytoplasmic determinants collectively termed germ plasm have been shown to play an essential role in the development of primordial germ cells (PGCs). Here, we report the identification of a nanos-like (nanos1) gene, which is expressed in the germ plasm and in the PGCs of the zebrafish. We find that several mechanisms act in concert to restrict the activity of Nanos1 to the germ cells including RNA localization and control over the stability and translatability of the RNA. Reducing the level of Nanos1 in zebrafish embryos revealed an essential role for the protein in ensuring proper migration and survival of PGCs in this vertebrate model organism.

Sequence-specific transactivation of the Drosophila twist gene by the dorsal gene product.

The maternal gene dorsal encodes a nuclear protein acting as a morphogen that determines the size and fate of regions along the dorsal-ventral axis of the Drosophila embryo. From previous genetic and biochemical studies it was hypothesized that dorsal might be responsible for the activation of the zygotic gene twist. In this report, regulatory sequences required for correct spatial and quantitative expression of twist are defined, by using phenotypic rescue and studying twist-beta-galactosidase expression. In addition, by transient cotransfection assays, we show that the dorsal protein specifically activates expression from the twist promoter. We demonstrate that dorsal is a sequence-specific DNA-binding protein that recognizes a motif similar to that recognized by the mammalian transcriptional activator NF-kappa B.

Expression of snail2, a second member of the zebrafish snail family, in cephalic mesendoderm and presumptive neural crest of wild-type and spadetail mutant embryos.

Transcripts of a newly discovered gene called snail2, encoding a zinc finger protein of the Snail family, first appear in rows of cephalic mesendodermal cells in gastrulating zebrafish embryos. At the end of gastrulation, snail2 RNA accumulates in a domain of ectodermal cells that mark the border between the epidermal epithelium and the neural plate and includes precursors of the neural crest. During somitogenesis, snail2 expression becomes restricted to neural crest. snail2 is thus one of the earliest genes yet known to be specifically expressed in neural crest in zebrafish embryos. Since snail2 is expressed in mesendoderm, a tissue layer whose convergence in the trunk is known to be altered in embryos homozygous for the spadetail mutation, we examined snail2 expression in spadetail embryos. In these mutants, the number of cephalic mesendodermal cells expressing snail2 is strongly reduced and the distribution of cells containing snail2 and no tail transcripts in the axial mesoderm is much broader than normal Moreover, the embryos are shorter than normal at the end of gastrulation. This shows that, in addition to the failure of paraxial mesoderm to converge normally in the trunk during gastrulation, spadetail also affects the elongation of the embryo and the convergence of axial and lateral mesendoderm in both trunk and head.

Novel FGF receptor (Z-FGFR4) is dynamically expressed in mesoderm and neurectoderm during early zebrafish embryogenesis.

We have identified a novel FGF receptor, Z-FGFR4, in zebrafish embryos. Z-FGFR4 is closely related to both chicken FREK (Marcelle et al. [1994] Development 120:683-694) and the Pleurodeles cDNA clone Pw-FGFR4 (also named PFR4). The Z-FGFR4 cDNA clones contain consensus sequences for two groups of two Ig-like domains, separated by eight acidic residues referred to as the "acid box." Z-FGFR4, therefore, is the first FGFR molecule yet described in vertebrates that contains four Ig domains in its amino-terminal region. Whole-mount in situ hybridization of staged zebrafish embryos, using probes prepared from a variety of domains of the Z-FGFR4 cDNA, reveal complex temporal and spatial expression patterns. Expression of Z-FGFR4 mRNA is first detected in embryos prior to gastrulation and then appears in prechordal plate mesendoderm. At this time, Z-FGFR mRNA is expressed in the epiblast in two distinct stripes which ultimately contribute to the brain. Eventually Z-FGFR4 transcripts are observed in forebrain, anterior hindbrain (rhombomeres 1, 3), and caudal hindbrain (rhombomere 7), as well as in the dorsal-most portion of the rostral spinal cord. Expression in axial mesendoderm appears transiently in notochord and segmental plate mesoderm. Eventually, Z-FGFR4 mRNA becomes restricted to the posterior somites and is absent in differentiated notochord. These detailed expression studies provide the basis for understanding FGFR function through an analysis, currently in progress, of the developmental consequences of Z-FGFR4 misexpression.

The lefty-related factor Xatv acts as a feedback inhibitor of nodal signaling in mesoderm induction and L-R axis development in xenopus.

In mouse, lefty genes play critical roles in the left-right (L-R) axis determination pathway. Here, we characterize the Xenopus lefty-related factor antivin (Xatv). Xatv expression is first observed in the marginal zone early during gastrulation, later becoming restricted to axial tissues. During tailbud stages, axial expression resolves to the neural tube floorplate, hypochord, and (transiently) the notochord anlage, and is joined by dynamic expression in the left lateral plate mesoderm (LPM) and left dorsal endoderm. An emerging paradigm in embryonic patterning is that secreted antagonists regulate the activity of intercellular signaling factors, thereby modulating cell fate specification. Xatv expression is rapidly induced by dorsoanterior-type mesoderm inducers such as activin or Xnr2. Xatv is not an inducer itself, but antagonizes both Xnr2 and activin. Together with its expression pattern, this suggests that Xatv functions during gastrulation in a negative feedback loop with Xnrs to affect the amount and/or character of mesoderm induced. Our data also provide insights into the way that lefty/nodal signals interact in the initiation of differential L-R morphogenesis. Right-sided misexpression of Xnr1 (endogenously expressed in the left LPM) induces bilateral Xatv expression. Left-sided Xatv overexpression suppresses Xnr1/XPitx2 expression in the left LPM, and leads to severely disturbed visceral asymmetry, suggesting that active 'left' signals are critical for L-R axis determination in frog embryos. We propose that the induction of lefty/Xatv in the left LPM by nodal/Xnr1 provides an efficient self-regulating mechanism to downregulate nodal/Xnr1 expression and ensure a transient 'left' signal within the embryo.

Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.

Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.