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Fetal Alcohol Spectrum Disorders (FASD) are a collection of disorders resulting from fetal ethanol exposure, which causes a wide range of physical, neurological and behavioral deficits including heightened susceptibility for alcoholism and addictive disorders. While a number of mechanisms have been proposed for how ethanol exposure disrupts brain development, with selective groups of neurons undergoing reduced proliferation, dysfunction and death, the induction of a new neurotransmitter phenotype by ethanol exposure has not yet been reported.The effects of embryonic and larval ethanol exposure on brain development were visually monitored using transgenic zebrafish expressing cell-specific green fluorescent protein (GFP) marker genes. Specific subsets of GFP-expressing neurons were highly sensitive to ethanol exposure, but only during defined developmental windows. In the med12 mutant, which affects the Mediator co-activator complex component Med12, exposure to lower concentrations of ethanol was sufficient to reduce GFP expression in transgenic embryos. In transgenic embryos and larva containing GFP driven by an oxytocin-like (oxtl) promoter, ethanol exposure dramatically up-regulated GFP expression in a small group of hindbrain neurons, while having no effect on expression in the neuroendocrine preoptic area.Alcohol exposure during limited embryonic periods impedes the development of specific, identifiable groups of neurons, and the med12 mutation sensitizes these neurons to the deleterious effects of ethanol. In contrast, ethanol exposure induces oxtl expression in the hindbrain, a finding with profound implications for understanding alcoholism and other addictive disorders.

Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.

In the vertebrate embryo, the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. In this study, we investigated the role of Lhx1 in specification of the kidney field by either overexpressing or depleting lhx1 in Xenopus embryos or depleting lhx1 in an explant culture system. By overexpressing a constitutively-active form of Lhx1, we established its capacity to expand the kidney field during the specification stage of kidney organogenesis. In addition, the ability of Lhx1 to expand the kidney field diminishes as kidney organogenesis transitions to the morphogenesis stage. In a complimentary set of experiments, we determined that embryos depleted of lhx1, show an almost complete loss of the kidney field. Using an explant culture system to induce kidney tissue, we confirmed that expression of genes from both proximal and distal kidney structures is affected by the absence of lhx1. Taken together our results demonstrate an essential role for Lhx1 in driving specification of the entire kidney field from the intermediate mesoderm.

Rhombomere boundaries form during hindbrain segmentation and are critical for maintaining segmental integrity and regulating migration in the hindbrain. Some genetic models affecting hindbrain boundary formation have been described, but involvement of components of the transcriptional mediator complex in boundary formation has not reported so far.The kto/med12 mutant zebrafish, which affects the Mediator component Med12, causes specific loss of rhombomere boundary cells even though segmentation of the hindbrain takes place at least in part. In kto mutant embryos, cells forming rhombomere boundaries were largely absent as indicated by the use of several marker genes. While no obvious increase in cell death was observed, we found a notable reduction of cell proliferation in the hindbrain of kto mutant zebrafish.The kto/med12 mutation results in specific defects of boundary cell formation in the zebrafish hindbrain.

Among Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role of zebrafish Myc genes.We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain.Mych is a novel factor required for neural crest cell survival in zebrafish.

Alpha 2 Macroglobulin family members have been studied extensively with respect to their roles in physiology and human disease including innate immunity and Alzheimer's disease, but little is known about a possible role in liver development loss-of-function in model systems.We report the isolation of the zebrafish alpha2 macroglobulin-like (A2ML) gene and its specific expression in the liver during differentiation. Morpholino-based knock-down of A2ML did not block the initial formation of the liver primordium, but inhibited liver growth and differentiation.This report on A2ML function in zebrafish development provides the first evidence for a specific role of an A2M family gene in liver formation during early embryogenesis in a vertebrate.

The analysis of the influence of fins on the flow and heat/mass transfer features for the impingement / effusion cooling with crossflow is described and a comparison is done with the cases without fins. Results show that the installation of fins significantly alters the flow and heat/mass transfer characteristics.

Background: Fetal Alcohol Spectrum Disorders (FASD) are a collection of disorders resulting from fetal ethanol exposure, which causes a wide range of physical, neurological and behavioral deficits including heightened susceptibility for alcoholism and addictive disorders. While a number of mechanisms have been proposed for how ethanol exposure disrupts brain development, with selective groups of neurons undergoing reduced proliferation, dysfunction and death, the induction of a new neurotransmitter phenotype by ethanol exposure has not yet been reported. Principal Findings: The effects of embryonic and larval ethanol exposure on brain development were visually monitored using transgenic zebrafish expressing cell-specific green fluorescent protein (GFP) marker genes. Specific subsets of GFP- expressing neurons were highly sensitive to ethanol exposure, but only during defined developmental windows. In the med12 mutant, which affects the Mediator co-activator complex component Med12, exposure to lower concentrations of ethanol was sufficient to reduce GFP expression in transgenic embryos. In transgenic embryos and larva containing GFP driven by an oxytocin-like (oxtl) promoter, ethanol exposure dramatically up-regulated GFP expression in a small group of hindbrain neurons, while having no effect on expression in the neuroendocrine preoptic area. Conclusions: Alcohol exposure during limited embryonic periods impedes the development of specific, identifiable groups of neurons, and the med12 mutation sensitizes these neurons to the deleterious effects of ethanol. In contrast, ethanol exposure induces oxtl expression in the hindbrain, a finding with profound implications for understanding alcoholism and other addictive disorders. [ABSTRACT FROM AUTHOR]

Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases [ABSTRACT FROM AUTHOR]

In the vertebrate embryo, the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. In this study, we investigated the role of Lhx1 in specification of the kidney field by either overexpressing or depleting lhx1 in Xenopus embryos or depleting lhx1 in an explant culture system. By overexpressing a constitutively-active form of Lhx1, we established its capacity to expand the kidney field during the specification stage of kidney organogenesis. In addition, the ability of Lhx1 to expand the kidney field diminishes as kidney organogenesis transitions to the morphogenesis stage. In a complimentary set of experiments, we determined that embryos depleted of lhx1, show an almost complete loss of the kidney field. Using an explant culture system to induce kidney tissue, we confirmed that expression of genes from both proximal and distal kidney structures is affected by the absence of lhx1. Taken together our results demonstrate an essential role for Lhx1 in driving specification of the entire kidney field from the intermediate mesoderm. [ABSTRACT FROM AUTHOR]

Angiogenesis plays an important role in vertebrate development and tumor growth. In this process, gicerin, which is known as a kind of cell adhesion molecule, has recently been reported to play an important role but its in vivo function is still unclear in developing vasculature. To address this issue, we used gain-of-function and loss-of-function analyses of gicerin in zebrafish. In the gain of function experiments using enforced expression of various domains of gicerin constructs, extracellular domain induced angiogenic sprouting defects, most notably in the intersegmental vessels, whereas the cytoplasmic domain of gicerin did not affect angiogenic sprouting. Moreover, morpholino-mediated knockdown of gicerin in embryos resulted in angiogenic sprouting defects in intersegmental vessels. Mechanistically, the angiogenic function of gicerin was found to be genetically linked to VEGF signaling in the knock-down experiments using vegf-a mRNA, VEGFR inhibitor and gicerin morpholino. In addition to the physiological angiogenesis during development, gicerin morphants efficiently blocked the tumor angiogenesis in zebrafish. Thus, knock-down of gicerin might have an important implication in controlling tumor angiogenesis. [ABSTRACT FROM AUTHOR]

Background: Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL). Results: We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent. Conclusion: We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pregastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning. [ABSTRACT FROM AUTHOR]

Establishment of left-right asymmetry in vertebrates requires nodal. Wnt-PCP and FGF signaling and involves ciliogenesis in a laterality organ. Effector genes through which FGF signaling affects laterality have not been described. We isolated the zebrafish ier2 and fibp1 genes as FGF target genes and show that their protein products interact. Knock down of these factors interferes with establishment of organ laterality and causes defective cilia formation in Kupffer's Vesicle. the zebrafish laterality organ. Cilia are also lost after suppression of FGF8. but can be rescued by injection of ier2 and fibp1 mRNA. We conclude that Ier2 and Fibp1 mediate FGF signaling in ciliogenesis in Kupffer's Vesicle and in the establishment of laterality in the zebrafish embryo. [ABSTRACT FROM AUTHOR]

A major goal for developmental biologists is to define the behaviors and molecular contents of differentiating cells. We have devised a strategy for isolating cells from diverse embryonic regions and stages in the zebrafish, using computer-guided laser photoconversion of injected Kaede protein and flow cytometry. This strategy enabled us to perform a genome-wide transcriptome comparison of germ layer precursor cells. Mesendoderm and ectoderm precursors cells isolated by this method differentiated appropriately in transplantation assays. Microarray analysis of these cells reidentified known genes at least as efficiently as previously reported strategies that relied on artificial mesendoderm activation or inhibition. We also identified a large set of uncharacterized mesendoderm-enriched genes as well as ectoderm-enriched genes. Loss-of-function studies revealed that one of these genes, the MAP kinase inhibitor dusp4, is essential for early development. Embryos injected with antisense morpholino oligonucleotides that targeted Dusp4 displayed necrosis of head tissues. Marker analysis during late gastrulation revealed a specific loss of sox17, but not of other endoderm markers, and analysis at later stages revealed a loss of foregut and pancreatic endoderm. This specific loss of sox17 establishes a new class of endoderm specification defect. [ABSTRACT FROM AUTHOR]

Mutation of the gene encoding the Mediator component thyroid hormone receptor-associated protein (TRAP)230/MED12 affects the development of multiple systems in zebrafish embryogenesis. We isolated two ethylnitrosourea-induced alleles in the gene encoding this protein and named the locus kohtalo (kto) after the homologous locus in Drosophila. Homozygous kto mutant zebrafish embryos show defects in brain, neural crest, and kidney development and die at ≈6 days postfertilization. In the affected tissues, differentiation is initiated and many cell type-specific genes are expressed, but there is a failure of morphogenesis and failure to complete differentiation. These results suggest that critical targets of TRAP230 function may include proteins important for cell mobility, cell sorting, and tissue assembly. [ABSTRACT FROM AUTHOR]

A complete zebrafish mespo cDNA encoding a protein of 131 amino acids with a bHLH domain in the C-terminal has been isolated. The bHLH domain of zebrafish Mespo is highly similar to those in the mouse, chick and Xenopus, sharing 82.4%, 80.4% and 74.5% amino acid identity, respectively. At 50% epiboly, the zebrafish mespo is first detected in the marginal zone of the blastoderm but excluding the prospective shield. Subsequently, mespo expression is intensified in the involuting mesoderm at 60% epiboly, and then restricted to the presomitic mesoderm (PSM) at 95% epiboly. At the 1-somite stage, mespo expression becomes reduced in the most rostral PSM. During segmentation, mespo expression is gradually downregulated at the most rostral segmental plate where cells are being coalesced to form somites. In spadetail mutant embryos, most of mespo-expressing cells were missing. [ABSTRACT FROM AUTHOR]

An abstract of the article "Embryonic Mesoderm and Endoderm Induction Requires the Actions of Non-Embryonic Nodal-Related Ligands and Mxtx2," by Sung-Kook Hong and colleagues is presented.

The present paper has investigated the effects of fins on the flow and heat/mass transfer characteristics for the impingement/effusion cooling with crossflow. The circular or rectangular fins are installed between two perforated plates, and the crossflow occurs between these two plates. The crossflow blowing ratio is varied from 0.5 to 1.5 for a fixed jet Reynolds number of 10,000. A naphthalene sublimation method is used to obtain local heat/mass transfer coefficients on the effusion plate. A numerical calculation is also performed to investigate the flow characteristics. The flow and heat/mass transfer characteristics are changed significantly due to the installation of fins. In the injection hole region, the wall jet spreads more widely than in the case without fins because the fins prevent the wall jet from being swept away by the crossflow. In the effusion hole region, a higher heat/mass transfer coefficient is obtained due to the flow disturbance and acceleration by the fin. As the blowing ratio increases, the effect of fins against the crossflow becomes more significant and subsequently the higher average heat/mass transfer coefficients are obtained. In particular, the cases with rectangular fins show an approximately 40-45% enhancement at the high blowing ratio of M=1.5. However, the increase in the blockage effect results in increased pressure loss in the channel. [ABSTRACT FROM AUTHOR]