Combined Pituitary Hormone Deficiency in lhx4-Knockout Zebrafish.

LIM homeobox 4 (LHX4) is a transcription factor crucial for anterior pituitary (AP) development. Patients with LHX4 mutation suffer from combined pituitary hormone deficiency (CPHD), short statures, reproductive and metabolic disorders and lethality in some cases. Lhx4-knockout (KO) mice fail to develop a normal AP and die shortly after birth. Here, we characterize a zebrafish lhx4-KO model to further investigate the importance of LHX4 in pituitary gland development and regulation. At the embryonic and larval stages, these fish express lower levels of tshb mRNA compared with their wildtype siblings. In adult lhx4-KO fish, the expressions of pituitary hormone-encoding transcripts, including growth hormone (gh), thyroid stimulating hormone (tshb), proopiomelanocortin (pomca) and follicle stimulating hormone (fshb), are reduced, the pomca promoter-driven expression in corticotrophs is dampened and luteinizing hormone (lhb)-producing gonadotrophs are severely depleted. In contrast to Lhx4-KO mice, Lhx4-deficient fish survive to adulthood, but with a reduced body size. Importantly, lhx4-KO males reach sexual maturity and are reproductively competent, whereas the females remain infertile with undeveloped ovaries. These phenotypes, which are reminiscent of those observed in CPHD patients, along with the advantages of the zebrafish for developmental genetics research, make this lhx4-KO fish an ideal vertebrate model to study the outcomes of LHX4 mutation.

Neuron-Derived Neurotrophic Factor Is Mutated in Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 × 10-6). Three heterozygous PTVs (p.Lys62∗, p.Tyr128Thrfs∗55, and p.Trp469∗, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.

Glial cells expressing visual cycle genes are vital for photoreceptor survival in the zebrafish pineal gland.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.

Genetic ablation of the Bsx homeodomain transcription factor in zebrafish: Impact on mature pineal gland morphology and circadian behavior.

The pineal gland is a neuroendocrine structure in the brain, which produces and secretes the hormone melatonin at nighttime and is considered a key element in the circadian clock system. Early morphogenesis of the gland is controlled by a number of transcription factors, some of which remain active in adult life. One of these is the brain-specific homeobox (Bsx), a highly conserved homeodomain transcription factor with a developmental role in the pineal gland of several species, including zebrafish, and regulatory roles in mature pinealocytes of the rat. To determine the role of Bsx in circadian biology, we here examined the effects of a bsx loss-of-function mutation on the pineal gland in adult zebrafish and on behavioral circadian rhythms in larvae. In pineal cell type-specific Gfp/Egfp reporter zebrafish lines, we did not detect fluorescence signals in the pineal area of homozygous (bsx-/- ) mutants. Interestingly, a nonpigmented area on the dorsal surface of the head above the gland, known as the pineal window, was pigmented in the homozygous mutants. Furthermore, a structure corresponding to the pineal gland was not detectable in the midline of the adult brain in histological sections analyzed by Nissl staining and S-antigen immunohistochemistry. Moreover, the levels of pineal transcripts were greatly reduced in bsx-/- mutants, as revealed by quantitative real-time polymerase chain reaction analysis. Notably, analysis of locomotor activity at the larval stage revealed altered circadian rhythmicity in the bsx mutants with periods and phases similar to wildtype, but severely reduced amplitudes in locomotor activity patterns. Thus, Bsx is essential for full development of the pineal gland, with its absence resulting in a phenotype of morphological pineal gland ablation and disrupted circadian behavior.

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day-night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator's period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep-wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Resource: A multi-species multi-timepoint transcriptome database and webpage for the pineal gland and retina.

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.

Ectopic over expression of kiss1 may compensate for the loss of kiss2.

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary-gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques-clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)-were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Interference with the Cannabinoid Receptor CB1R Results in Miswiring of GnRH3 and AgRP1 Axons in Zebrafish Embryos.

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway.

Gne depletion during zebrafish development impairs skeletal muscle structure and function.

GNE Myopathy is a rare recessively inherited neuromuscular disorder caused by mutations in the GNE gene, which codes for the key enzyme in the metabolic pathway of sialic acid synthesis. The process by which GNE mutations lead to myopathy is not well understood. By in situ hybridization and gne promoter-driven fluorescent transgenic fish generation, we have characterized the spatiotemporal expression pattern of the zebrafish gne gene and have shown that it is highly conserved compared with the human ortholog. We also show the deposition of maternal gne mRNA and maternal GNE protein at the earliest embryonic stage, emphasizing the critical role of gne in embryonic development. Injection of morpholino (MO)-modified antisense oligonucleotides specifically designed to knockdown gne, into one-cell embryos lead to a variety of phenotypic severity. Characterization of the gne knockdown morphants showed a significantly reduced locomotor activity as well as distorted muscle integrity, including a reduction in the number of muscle myofibers, even in mild or intermediate phenotype morphants. These findings were further confirmed by electron microscopy studies, where large gaps between sarcolemmas were visualized, although normal sarcomeric structures were maintained. These results demonstrate a critical novel role for gne in embryonic development and particularly in myofiber development, muscle integrity and activity.

Knockdown of unc119c results in visual impairment and early-onset retinal dystrophy in zebrafish.

PURPOSE: UNC119 proteins are involved in G protein trafficking in mouse retinal photoreceptors and Caenorhabditis elegans olfactory neurons. An Unc119 null allele is associated with cone-rod dystrophy in mouse, but the mechanism leading to disease is not understood. We studied the role of Unc119 paralogs and Arl3l2 in zebrafish vision and retinal organization resulting from unc119c and arl3l2 knockdown. METHODS: Zebrafish unc119c was amplified by PCR from retina and pineal gland cDNA. Its expression pattern in the eye and pineal gland was determined by whole-mount in-situ hybridization. unc119c and arl3l2 were knocked down using morpholino-modified oligonucleotides (MO). Their visual function was assessed with a quantitative optomotor assay on 6 days post-fertilization larvae. Retinal morphology was analyzed using immunohistochemistry with anti-cone arrestin (zpr-1) and anti-cone transducin-α (GNAT2) antibodies. RESULTS: The zebrafish genome contains four genes encoding unc119 paralogs located on different chromosomes. The exon/intron arrangements of these genes are identical. Three Unc119 paralogs are expressed in the zebrafish retina, termed Unc119a-c. Based on sequence similarity, Unc119a and Unc119b are orthologs of mammalian UNC119a and UNC119b, respectively. A third, Unc119c, is unique and not present in mammals. Whole mount in-situ hybridization revealed that unc119a and unc119b RNA are ubiquitously expressed in the CNS, and unc119c is specifically expressed in photoreceptive tissues (pineal gland and retina). A Unc119 interactant, Arl3l2 also localizes to the pineal gland and the retina. As measured by the optomotor response, unc119c and arl3l2 knockdown resulted in significantly lower vision compared to wild-type zebrafish larvae and control morpholino (MO). Immunohistological analysis with anti-cone transducin and anti-cone arrestin (zpr-1) indicates that knockdown of unc119c leads to photoreceptor degeneration mostly affecting cones. CONCLUSIONS: Our results suggest that Unc119c is the only Unc119 paralog that is highly specific to the retina in zebrafish. Unc119c and Arl3l2 proteins are important for the function of cones.

The light-induced transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian clockwork by light.

Light constitutes a primary signal whereby endogenous circadian clocks are synchronized ('entrained') with the day/night cycle. The molecular mechanisms underlying this vital process are known to require gene activation, yet are incompletely understood. Here, the light-induced transcriptome in the zebrafish central clock organ, the pineal gland, was characterized by messenger RNA (mRNA) sequencing (mRNA-seq) and microarray analyses, resulting in the identification of multiple light-induced mRNAs. Interestingly, a considerable portion of the molecular clock (14 genes) is light-induced in the pineal gland. Four of these genes, encoding the transcription factors dec1, reverbb1, e4bp4-5 and e4bp4-6, differentially affected clock- and light-regulated promoter activation, suggesting that light-input is conveyed to the core clock machinery via diverse mechanisms. Moreover, we show that dec1, as well as the core clock gene per2, is essential for light-entrainment of rhythmic locomotor activity in zebrafish larvae. Additionally, we used microRNA (miRNA) sequencing (miR-seq) and identified pineal-enhanced and light-induced miRNAs. One such miRNA, miR-183, is shown to downregulate e4bp4-6 mRNA through a 3'UTR target site, and importantly, to regulate the rhythmic mRNA levels of aanat2, the key enzyme in melatonin synthesis. Together, this genome-wide approach and functional characterization of light-induced factors indicate a multi-level regulation of the circadian clockwork by light.

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system.

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5(-/-) olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5-Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Subfunctionalization of arylalkylamine N-acetyltransferases in the sea bass Dicentrarchus labrax: two-ones for one two.

Melatonin is an important component of the vertebrates circadian system, synthetized from serotonin by the successive action of the arylalkylamine N-acetyltransferase (Aanat: serotonin→N-acetylserotonin) and acetylserotonin-O-methyltransferase (Asmt: N-acetylserotonin→melatonin). Aanat is responsible for the daily rhythm in melatonin production. Teleost fish are unique because they express two Aanat genes, aanat1 and aanat2, mainly expressed in the retina and pineal gland, respectively. In silico analysis indicated that the teleost-specific whole-genome duplication generated Aanat1 duplicates (aanat1a and aanat1b); some fish express both of them, while others express either one of the isoforms. Here, we bring the first information on the structure, function, and distribution of Aanat1a and Aanat1b in a teleost, the sea bass Dicentrarchus labrax. Aanat1a and Aanat1b displayed a wide and distinct distribution in the nervous system and peripheral tissues, while Aanat2 appeared as a pineal enzyme. Co-expression of Aanats with asmt was found in the pineal gland and the three retinal nuclear layers. Enzyme kinetics indicated subtle differences in the affinity and catalytic efficiency of Aanat1a and Aanat1b for indolethylamines and phenylethylamines, respectively. Our data are consistent with the idea that Aanat2 is a pineal enzyme involved in melatonin production, while Aanat1 enzymes have a broader range of functions including melatonin synthesis in the retina, and catabolism of serotonin and dopamine in the retina and other tissues. The data are discussed in light of the recently uncovered roles of N-acetylserotonin and N-acetyldopamine as antioxidants, neuroprotectants, and modulators of cell proliferation and enzyme activities.

Systematic identification of rhythmic genes reveals camk1gb as a new element in the circadian clockwork.

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA-seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.

Novel evolutionary-conserved role for the activity-dependent neuroprotective protein (ADNP) family that is important for erythropoiesis.

BACKGROUND: ADNP is vital for embryonic development. Is this function conserved for the homologous protein ADNP2? RESULTS: Down-regulation/silencing of ADNP or ADNP2 in zebrafish embryos or mouse erythroleukemia cells inhibited erythroid maturation, with ADNP directly associating with the ß-globin locus control region. CONCLUSION: ADNPs are novel molecular regulators of erythropoiesis. SIGNIFICANCE: New regulators of globin synthesis are suggested. Activity-dependent neuroprotective protein (ADNP) and its homologue ADNP2 belong to a homeodomain, the zinc finger-containing protein family. ADNP is essential for mouse embryonic brain formation. ADNP2 is associated with cell survival, but its role in embryogenesis has not been evaluated. Here, we describe the use of the zebrafish model to elucidate the developmental roles of ADNP and ADNP2. Although we expected brain defects, we were astonished to discover that the knockdown zebrafish embryos were actually lacking blood and suffered from defective hemoglobin production. Evolutionary conservation was established using mouse erythroleukemia (MEL) cells, a well studied erythropoiesis model, in which silencing of ADNP or ADNP2 produced similar results as in zebrafish. Exogenous RNA encoding ADNP/ADNP2 rescued the MEL cell undifferentiated state, demonstrating phenotype specificity. Brg1, an ADNP-interacting chromatin-remodeling protein involved in erythropoiesis through regulation of the globin locus, was shown here to interact also with ADNP2. Furthermore, chromatin immunoprecipitation revealed recruitment of ADNP, similar to Brg1, to the mouse ß-globin locus control region in MEL cells. This recruitment was apparently diminished upon dimethyl sulfoxide (DMSO)-induced erythrocyte differentiation compared with the nondifferentiated state. Importantly, exogenous RNA encoding ADNP/ADNP2 significantly increased ß-globin expression in MEL cells in the absence of any other differentiation factors. Taken together, our results reveal an ancestral role for the ADNP protein family in maturation and differentiation of the erythroid lineage, associated with direct regulation of ß-globin expression.

Establishment of cell lines from individual zebrafish embryos.

With the increasing use of fish as model species for research, cell cultures derived from caudal fin explants as well as pre-hatching stage embryos have provided powerful in vitro tools that can complement or serve as an ethically more acceptable alternative to live animal experiments. The widely-used protocols to establish these lines require, as a starting point, homogeneous pools of embryos or viable adult fish which are large enough for collecting sufficient fin tissue. This excludes the use of fish lines with adverse phenotypes or lines that exhibit mortality at early developmental stages and so can only be propagated as heterozygotes. Specifically, when no visually overt mutant phenotype is detectable for identifying homozygous mutants at early embryonic stages, it is then impossible to sort pools of embryos with the same genotypes to generate cell lines from the progeny of a heterozygote in-cross. Here, we describe a simple protocol to generate cell lines on a large scale starting from individual early embryos that can subsequently be genotyped by polymerase chain reaction. This protocol should help to establish fish cell culture models as a routine approach for the functional characterization of genetic changes in fish models such as the zebrafish. Furthermore, it should contribute to a reduction of experiments which are ethically discouraged to avoid pain and distress.

Light directs zebrafish period2 expression via conserved D and E boxes.

For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.