Identification of transcripts potentially involved in neural tube closure using RNA sequencing.

Anencephaly is a fatal human neural tube defect (NTD) in which the anterior neural tube remains open. Zebrafish embryos with reduced Nodal signaling display an open anterior neural tube phenotype that is analogous to anencephaly. Previous work from our laboratory suggests that Nodal signaling acts through induction of the head mesendoderm and mesoderm. Head mesendoderm/mesoderm then, through an unknown mechanism, promotes formation of the polarized neuroepithelium that is capable of undergoing the movements required for closure. We compared the transcriptome of embryos treated with a Nodal signaling inhibitor at sphere stage, which causes NTDs, to embryos treated at 30% epiboly, which does not cause NTDs. This screen identified over 3,000 transcripts with potential roles in anterior neurulation. Expression of several genes encoding components of tight and adherens junctions was significantly reduced, supporting the model that Nodal signaling regulates formation of the neuroepithelium. mRNAs involved in Wnt, FGF, and BMP signaling were also differentially expressed, suggesting these pathways might regulate anterior neurulation. In support of this, we found that pharmacological inhibition of FGF-receptor function causes an open anterior NTD as well as loss of mesodermal derivatives. This suggests that Nodal and FGF signaling both promote anterior neurulation through induction of head mesoderm.

Temporal and spatial requirements for Nodal-induced anterior mesendoderm and mesoderm in anterior neurulation.

Zebrafish with defective Nodal signaling have a phenotype analogous to the fatal human birth defect anencephaly, which is caused by an open anterior neural tube. Previous work in our laboratory found that anterior open neural tube phenotypes in Nodal signaling mutants were caused by lack of mesendodermal/mesodermal tissues. Defects in these mutants are already apparent at neural plate stage, before the neuroepithelium starts to fold into a tube. Consistent with this, we found that the requirement for Nodal signaling maps to mid-late blastula stages. This timing correlates with the timing of prechordal plate mesendoderm and anterior mesoderm induction, suggesting these tissues act to promote neurulation. To further identify tissues important for neurulation, we took advantage of the variable phenotypes in Nodal signaling-deficient sqt mutant and Lefty1-overexpressing embryos. Statistical analysis indicated a strong, positive correlation between a closed neural tube and presence of several mesendoderm/mesoderm-derived tissues (hatching glands, cephalic paraxial mesoderm, notochord, and head muscles). However, the neural tube was closed in a subset of embryos that lacked any one of these tissues. This suggests that several types of Nodal-induced mesendodermal/mesodermal precursors are competent to promote neurulation.

Failure in closure of the anterior neural tube causes left isomerization of the zebrafish epithalamus.

Differences between the left and right sides of the brain are present in many animal species. For instance, in humans the left cerebral hemisphere is largely responsible for language and tool use and the right for processing spatial information. Zebrafish have prominent left-right asymmetries in their epithalamus that have been associated with differential left and right eye use and navigational behavior. In wild-type (WT) zebrafish embryos, Nodal pathway genes are expressed in the left side of the pineal anlage. Shortly thereafter, a parapineal organ forms to the left of the pineal. The parapineal organ causes differences in gene expression, neuropil density, and connectivity of the left and right habenula nuclei. In embryos that have an open neural tube, such as embryos that are deficient in Nodal signaling or the cell adhesion protein N-cadherin, the left and right sides of the developing epithalamus remain separated from one another. We find that the brains of these embryos often become left isomerized: both sides of the brain develop morphology and gene expression patterns that are characteristic of the left side. However, other aspects of epithalamic development, such as differentiation of specific neuronal cell types, are intact. We propose that there is a mechanism in embryos with closed neural tubes that prevents both sides from developing like the left side. This mechanism fails when the two sides of the epithalamus are widely separated from one another, suggesting that it is dependent upon a signaling protein with limited range.

Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development.

Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafish's transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.

Otx5 regulates genes that show circadian expression in the zebrafish pineal complex.

The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus-the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.

Changes in basic psychological needs, passion, and well-being of first-semester graduate students.

OBJECTIVE: Examine changes in graduate student health and well-being in the first semester. PARTICIPANTS: Full-time, first-semester graduate students (N = 74) from a midsized midwestern university. METHOD: Graduate students were surveyed prior to starting their master's program and 10 weeks later. Passion for academics, basic psychological needs, physical and mental health symptoms, positive and negative affects, and quality of life were assessed. RESULTS: Need satisfaction, harmonious passion, and indicators of well-being decreased across the first semester, whereas need frustration and indicators of ill-being increased over the first semester. Obsessive passion, harmonious passion, need satisfaction, and need frustration were associated with students' well-being at the end of the semester, with need frustration being the most robust predictor. CONCLUSIONS: Although most graduate students reported good general health and moderately low mental health symptoms, findings suggest that a need supportive environment may contribute to better health and well-being.

Illustrated Protocols to Improve Undergraduate Student Research Independence.

One big challenge for undergraduate research students is gaining independence in the laboratory. In this curricular project, undergraduate students transformed research protocols developed for experienced scientists into protocols understandable to someone new to a laboratory. This process enabled themselves and other students to more quickly learn and master new techniques and advance to independent projects. Typically, students started with an original research protocol that assumed basic knowledge, such as instructions that came with a kit (i.e. plasmid purification kit instructions). Students created notes that explained the purpose of each step and reagent and provided example calculations. Then students illustrated the protocols with photos of materials needed, equipment used, action shots of difficult steps and screenshots of software programs. This approach has been used by students in laboratory courses and by new independent research students learning laboratory techniques. In the laboratory courses where students contributed to this project as part of a writing assignment, additional professional experience was gained by presenting a talk about their completed Illustrated Protocols to their classmates and by creating group posters that were presented at an undergraduate research symposium. After completion of this activity, undergraduate students gained confidence by applying their new knowledge to create user-friendly protocols. Students reported increased understanding of what is happening in each step, while instructors reported increased student independence and confidence that the protocol was being applied correctly and consistently. Thus, designing Illustrated Protocols enhanced learning and independence for the students creating the protocol and provided valuable help for future students.

Circadian rhythms in the pineal organ persist in zebrafish larvae that lack ventral brain.

BACKGROUND: The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish cyclops (cyc) mutants lack ventral brain, including the region that gives rise to the SCN. We have used cyc embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression. RESULTS: We find that pineal rhythms are present in cyc mutants despite the absence of an SCN. The arginine vasopressin-like protein (Avpl, formerly called Vasotocin) is a peptide hormone expressed in and around the SCN. We find avpl mRNA is absent in cyc mutants, supporting previous work suggesting the SCN is missing. In contrast, expression of the putative circadian clock genes, cryptochrome 1b (cry1b) and cryptochrome 3 (cry3), in the brain of the developing fish is unaltered. Expression of two pineal rhythmic genes, exo-rhodopsin (exorh) and serotonin-N-acetyltransferase (aanat2), involved in photoreception and melatonin synthesis, respectively, is also similar between cyc embryos and their wildtype (WT) siblings. The timing of the peaks and troughs of expression are the same, although the amplitude of expression is slightly decreased in the mutants. Cyclic gene expression persists for two days in cyc embryos transferred to constant light or constant dark, suggesting a circadian clock is driving the rhythms. However, the amplitude of rhythms in cyc mutants kept in constant conditions decreased more quickly than in their WT siblings. CONCLUSION: Our data suggests that circadian rhythms can be initiated and maintained in the absence of SCN and other tissues in the ventral brain. However, the SCN may have a role in regulating the amplitude of rhythms when environmental cues are absent. This provides some of the first evidence that the SCN of teleosts is not essential for establishing circadian rhythms during development. Several SCN-independent circadian rhythms have also been found in mammalian species. Thus, zebrafish may serve as a model system for understanding how vertebrate embryos coordinate rhythms that are controlled by different circadian clocks.

Amyloid precursor protein is required for convergent-extension movements during Zebrafish development.

Amyloid precursor protein (APP) has been a focus of intense investigation because of its role in Alzheimer's disease (AD), however, its biological function remains uncertain. Loss of APP and APP-like proteins results in postnatal lethality in mice, suggesting a role during embryogenesis. Here we show that in a zebrafish model system, knock down of APP results in the generation of fish with dramatically reduced body length and a short, curly tail. In situ examination of gene expression suggests that the APP morphant embryos have defective convergent-extension movements. We also show that wild-type human APP rescues the morphant phenotype, but the Swedish mutant APP, which causes familial AD (fAD), does not rescue the developmental defects. Collectively, this work demonstrates that the zebrafish model is a powerful system to define the role of APP during embryonic development and to evaluate the functional activity of fAD mutant APP.

The CCN family member Wisp3, mutant in progressive pseudorheumatoid dysplasia, modulates BMP and Wnt signaling.

In humans, loss-of-function mutations in the gene encoding Wnt1 inducible signaling pathway protein 3 (WISP3) cause the autosomal-recessive skeletal disorder progressive pseudorheumatoid dysplasia (PPD). However, in mice there is no apparent phenotype caused by Wisp3 deficiency or overexpression. Consequently, the in vivo activities of Wisp3 have remained elusive. We cloned the zebrafish ortholog of Wisp3 and investigated its biologic activity in vivo using gain-of-function and loss-of-function approaches. Overexpression of zebrafish Wisp3 protein inhibited bone morphogenetic protein (BMP) and Wnt signaling in developing zebrafish. Conditioned medium-containing zebrafish and human Wisp3 also inhibited BMP and Wnt signaling in mammalian cells by binding to BMP ligand and to the Wnt coreceptors low-density lipoprotein receptor-related protein 6 (LRP6) and Frizzled, respectively. Wisp3 proteins containing disease-causing amino acid substitutions found in patients with PPD had reduced activity in these assays. Morpholino-mediated inhibition of zebrafish Wisp3 protein expression in developing zebrafish affected pharyngeal cartilage size and shape. These data provide a biologic assay for Wisp3, reveal a role for Wisp3 during zebrafish cartilage development, and suggest that dysregulation of BMP and/or Wnt signaling contributes to cartilage failure in humans with PPD.

Mathematical Analysis of Melanocyte Patterns on Danio rerio.

The study of zebrafish skin pattern development could lead to a better understanding of how these patterns are generated and how they evolved. To compare and contrast wild-type (WT) striped and leopardt1 mutant spotted patterns, photographs were taken of the developing fish. Initial observations led to the hypothesis that the black melanocyte spots in leopardt1 mutants were not randomly distributed, but rather were located in "dashed" stripes. To test this, melanocyte-spot-sized transparent grids were overlaid onto photographs and the location of melanocyte clusters was recorded. The grid maps were used to identify whether a black, melanocyte positive, grid area was present adjacent to each melanocyte cluster in each cardinal and intercardinal direction. In addition, Python-based computer programs were used to analyze the photographs at the pixel level. When analyzed using analysis of variance and logistic regression models, the striped and spotted patterns expressed more similarities than expected. In the leopardt1 zebrafish, the spots were organized into dashed stripes that had similar locations to the WT stripes. This research suggests that spotted and striped patterns are related. Further, the leopardt1 spots were farther apart along the dorsal-ventral axis than in the anterior-posterior direction, suggesting that different mechanisms control spacing along these two axes.

Cloning, localization, and functional expression of the electrogenic Na+ bicarbonate cotransporter (NBCe1) from zebrafish.

Mutations in the electrogenic Na+/nHCO3- cotransporter (NBCe1, SLC4A4) cause severe proximal renal tubular acidosis, glaucoma, and cataracts in humans, indicating NBCe1 has a critical role in acid-base homeostasis and ocular fluid transport. To better understand the homeostatic roles and protein ontogeny of NBCe1, we have cloned, localized, and downregulated NBCe1 expression in zebrafish, and examined its transport characteristics when expressed in Xenopus oocytes. Zebrafish NBCe1 (zNBCe1) is 80% identical to published mammalian NBCe1 cDNAs. Like other fish NBCe1 clones, zebrafish NBCe1 is most similar to the pancreatic form of mammalian NBC (Slc4a4-B) but appears to be the dominant isoform found in zebrafish. In situ hybridization of embryos demonstrated mRNA expression in kidney pronephros and eye by 24 h postfertilization (hpf) and gill and brain by 120 hpf. Immunohistochemical labeling demonstrated expression in adult zebrafish eye and gill. Morpholino knockdown studies demonstrated roles in eye and brain development and caused edema, indicating altered fluid and electrolyte balance. With the use of microelectrodes to measure membrane potential (Vm), voltage clamp (VC), intracellular pH (pH(i)), or intracellular Na+ activity (aNa(i)), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCe1s, demonstrating electrogenic Na+ and HCO3- transport as well as similar drug sensitivity, including inhibition by 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and tenidap. These data indicate that NBCe1 in zebrafish shares many characteristics with mammalian NBCe1, including tissue distribution, importance in systemic water and electrolyte balance, and electrogenic transport of Na+ and HCO3-. Thus zebrafish promise to be useful model system for studies of NBCe1 physiology.

Novel functions for Period 3 and Exo-rhodopsin in rhythmic transcription and melatonin biosynthesis within the zebrafish pineal organ.

Entrainment of circadian clocks to environmental cues such as photoperiod ensures that daily biological rhythms stay in synchronization with the Earth's rotation. The vertebrate pineal organ has a conserved role in circadian regulation as the primary source of the nocturnal hormone melatonin. In lower vertebrates, the pineal has an endogenous circadian clock as well as photoreceptive cells that regulate this clock. The zebrafish opsin protein Exo-rhodopsin (Exorh) is expressed in pineal photoreceptors and is a candidate to mediate the effects of environmental light on pineal rhythms and melatonin synthesis. We demonstrate that Exorh has an important role in regulating gene transcription within the pineal. In developing embryos that lack Exorh, expression of the exorh gene itself and of the melatonin synthesis gene serotonin N-acetyl transferase 2 (aanat2) are significantly reduced. This suggests that the Exorh protein at the cell membrane is part of a signaling pathway that positively regulates transcription of these genes, and ultimately melatonin production, in the pineal. Like many other opsin genes, exorh is expressed with a daily rhythm: mRNA levels are higher at night than during the day. We found that the transcription factor Orthodenticle homeobox 5 (Otx5) activates exorh transcription, while the putative circadian clock component Period 3 (Per3) represses expression during the day, thereby contributing to the rhythm of transcription. This work identifies novel roles for Exorh and Per3, and gives insight into potential interactions between the sensory and circadian systems within the pineal.

Nodal signaling is required for closure of the anterior neural tube in zebrafish.

BACKGROUND: Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish. RESULTS: We find that the neural tube in the presumptive forebrain fails to close in zebrafish Nodal signaling mutants. For instance, the cells that will give rise to the pineal organ fail to move from the lateral edges of the neural plate to the midline of the diencephalon. The open neural tube in Nodal signaling mutants may be due in part to reduced function of N-cadherin, a cell adhesion molecule expressed in the neural tube and required for neural tube closure. N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling. Further, N-cadherin mutants and morphants have a pineal phenotype similar to that of mutants with deficiencies in the Nodal pathway. Overexpression of an activated form of the TGFbeta Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling. We find that overexpression of Taram-A* also corrects their open neural tube defect. This suggests that, as in mammals, the mesoderm and endoderm have an important role in regulating closure of the anterior neural tube of zebrafish. CONCLUSION: This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain.

Leaning to the left: laterality in the zebrafish forebrain.

How the brain becomes lateralized is poorly understood. By contrast, much is known about molecular cues that specify the left-right axis of the body, fashioning the asymmetric morphology and positioning of the visceral organs. In zebrafish, the Nodal signaling pathway functions in visceral asymmetry and also in the embryonic brain, to bias laterality of the epithalamus. Formation of an asymmetric pineal complex differentially influences adjacent diencephalic nuclei, the left and right habenulae, which acquire distinctive molecular and cellular features. Results from the genetically tractable zebrafish system provide a promising entry point for exploring how left-right biases are established and propagated in the developing vertebrate brain.

Learning to Fish with Genetics: A Primer on the Vertebrate Model Danio rerio.

In the last 30 years, the zebrafish has become a widely used model organism for research on vertebrate development and disease. Through a powerful combination of genetics and experimental embryology, significant inroads have been made into the regulation of embryonic axis formation, organogenesis, and the development of neural networks. Research with this model has also expanded into other areas, including the genetic regulation of aging, regeneration, and animal behavior. Zebrafish are a popular model because of the ease with which they can be maintained, their small size and low cost, the ability to obtain hundreds of embryos on a daily basis, and the accessibility, translucency, and rapidity of early developmental stages. This primer describes the swift progress of genetic approaches in zebrafish and highlights recent advances that have led to new insights into vertebrate biology.

Learning the scientific method using GloFish.

Here we describe projects that used GloFish, brightly colored, fluorescent, transgenic zebrafish, in experiments that enabled students to carry out all steps in the scientific method. In the first project, students in an undergraduate genetics laboratory course successfully tested hypotheses about the relationships between GloFish phenotypes and genotypes using PCR, fluorescence microscopy, and test crosses. In the second and third projects, students doing independent research carried out hypothesis-driven experiments that also developed new GloFish projects for future genetics laboratory students. Brianna Vick, an undergraduate student, identified causes of the different shades of color found in orange GloFish. Adrianna Pollak, as part of a high school science fair project, characterized the fluorescence emission patterns of all of the commercially available colors of GloFish (red, orange, yellow, green, blue, and purple). The genetics laboratory students carrying out the first project found that learning new techniques and applying their knowledge of genetics were valuable. However, assessments of their learning suggest that this project was not challenging to many of the students. Thus, the independent projects will be valuable as bases to widen the scope and range of difficulty of experiments available to future genetics laboratory students.

Making a difference: education at the 10th International Conference on Zebrafish Development and Genetics.

Scientists, educators, and students met at the 10th International Conference on Zebrafish Development and Genetics during the 2-day Education Workshop, chaired by Dr. Jennifer Liang and supported in part by the Genetics Society of America. The goal of the workshop was to share expertise, to discuss the challenges faced when using zebrafish in the classroom, and to articulate goals for expanding the impact of zebrafish in education.