Zebrafish as a Model for Fetal Alcohol Spectrum Disorders

In this review, we will discuss zebrafish as a model for studying mechanisms of human fetal alcohol spectrum disorders (FASDs). We will overview the studies on FASDs so far and will discuss with specific focus on the mechanisms by which alcohol alters cell migration during the early embryogenesis including blastula, gastrula, and organogenesis stages which later cause morphological defects in the brain and other tissues. FASDs are caused by an elevated alcohol level in the pregnant mother’s body. The symptoms of FASDs include microcephaly, holoprosencephaly, craniofacial abnormalities, and cardiac defects with birth defect in severe cases, and in milder cases, the symptoms lead to developmental and learning disabilities. The transparent zebrafish embryo offers an ideal model system to investigate the genetic, cellular, and organismal responses to alcohol. In the zebrafish, the effects of alcohol were observed in many places during the embryo development from the stem cell gene expression at the blastula/gastrula stage, gastrulation cell movement, morphogenesis of the central nervous system, and neuronal development. The data revealed that ethanol suppresses convergence, extension, and epiboly cell movement at the gastrula stage and cause the failure of normal neural plate formation. Subsequently, other cell movements including neurulation, eye field morphogenesis, and neural crest migration are also suppressed, leading to the malformation of the brain and spinal cord, including microcephaly, cyclopia, spinal bifida, and craniofacial abnormalities. The testing cell migration in zebrafish would provide convenient biomarkers for the toxicity of alcohol and other related chemicals, and investigate the molecular link between the target signaling pathways, following brain development.

Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag

Background: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis. Results: To overcome these challenges, we adapted the chromatin profiling methods CUT&RUN and CUT&Tag for use in zebrafish, and applied these methods to generate high resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z from mid gastrula stage embryos. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences. Conclusions: Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis.

PERKEMBANGAN EMBRIO DAN PERFORMA AWAL LARVA TIGA SPESIES IKAN TOR INDONESIA

Tor fish is a potential local fish. It has social, economic and religious value and contains albumin which is equivalent to snake head fish. As a local fish cultivation potential, an accurate description of the early development stages of this species, will have value for ichthyologists and can facilitate cultivation efforts to increase production. This study aims to understand the embryology of three tor fish species and to determine the condition of larval performance at the beginning of hatching. The division process begins when the cell nucleus is divided into 2 blastomers, then continues to reach 32 cells in the first 10 hours after fertilization. At 10–20 hours after fertilization the eggs enter the morula, blastula and gastrula phases. After 20 hours, the organogenesis stage occurs. Tor douronensis hatched the fastest, more than 100 hours after fertilization (0.70–0.80 cm length, 0.0073 g weight and 8.40±1.83% abnormality). Tor soro over 120 hours (length 0.80–0.90 cm, weight 0.0125 g and abnormality 2.47±0.12%) and Tor tambroides above 140 hours after fertilization (length 1,00–1,09 cm, weight 0,0146 g and abnormality 2.93±0.31%). It can be concluded that there is no difference in the process of embryogenesis of the three species until the gastrula stage. The difference arises in the organogenesis phase, where the Tor douronensis organ develops most rapidly, followed by Tor soro and Tor tambroides.

Inhibition of ecto-5′-nucleotidase and adenosine deaminase is able to reverse long-term behavioural effects of early ethanol exposure in zebrafish (Danio rerio)

The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5′-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5′-nucleotidase and adenosine deaminase activities.

Nucleoplasmin is a limiting component in the scaling of nuclear size with cytoplasmic volume

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during Xenopus laevis embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula-stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics. Nuclei grew and reached new steady-state sizes as a function of cytoplasmic volume, supporting a limiting component mechanism of nuclear size control. Through biochemical fractionation, we identified the histone chaperone nucleoplasmin (Npm2) as a putative nuclear size effector. Cellular amounts of Npm2 decrease over development, and nuclear size was sensitive to Npm2 levels both in vitro and in vivo, affecting nuclear histone levels and chromatin organization. We propose that reductions in cell volume and the amounts of limiting components, such as Npm2, contribute to developmental nuclear size scaling.

Experimental hybridization of sympatric temnopleurid sea urchins living in Yamaguchi, Japan

Three sympatric species of temnopleurid sea urchins, Temopleurus toreumaticus (Leske, 1778), T. hardwickii (Gray, 1855) and Mespilia globulus (Linnaeus, 1758), occupy a habitat located a short distance from the shore of the Seto Inland Sea, Yamaguchi Prefecture, Japan. Breeding seasons overlap considerably among these species. These species exhibit species-specific differences in embryonic and larval development such as a wrinkled zygote, wrinkled blastula, archenteron invagination, larval skeleton and juvenile morphology. In this study, we determined whether interbreeding among these species is possible. Investigations revealed that fertilization succeeded with all mating combination patterns and that all fertilized eggs developed to the gastrula stage. The blastula and gastrula of hybrids formed in a manner similar to the maternal whereas the developmental delay observed followed the pattern of the paternal species T. toreumaticus fertilized at a greater proportion than other pairs and metamorphosed. These results suggest that eggs of this species may have weaker fertilization block in general. At the early developmental stages, hybrid embryos from T. toreumaticus mothers express maternal, while some hybrids derived from sperm of T. toreumaticus and the eggs of other species ceased development at the 4-armed larval stage. The hybrids of T. hardwickii and M. globulus ceased development at the gastrula stage, suggesting that these species have a greater degree of genetically isolation distance. In hybrids of T. toreumaticus and strongylocentrotid Hemicentrotus pulcherrimus (A. Agassiz, 1864), larval skeletons expressed features from both species. These results suggest that traits of hybrids are derived not only from one or both original species, but that a mosaic effect can be obtained depending on the traits. These results indicate that temnopleurids have the weaker fertilization block mechanisms. Isozyme analyses showed that hybrid prisms of T. toreumaticus eggs and M. globulus sperm possess enzymatic patterns of malate dehydrogenase derived from each parent, whereas the enzymatic pattern of glucose-6-phosphate dehydrogenase is derived from eggs, suggesting that malate dehydrogenase activity may be useful in detecting naturally occurring adult hybrids among these species in the Yamaguchi coastal area.

Embryonic development of Peruvian grunt Anisotremus scapularis (Perciformes: Haemulidae)

Peruvian grunt Anisotremus scapularis is distributed from Ecuador to Chile and it is considered an important aquaculture resource in Peru. Knowledge of embryonic development is crucial because it is part of the basic biology of a species. The aim of this study was to describe the embryonic stages of Peruvian grunt. The eggs were obtained by natural spawning and reared at 19 °C under laboratory conditions. Morphometric characteristics of the egg were evaluated: diameter (0.752 ± 0.025 mm) (mean ± sd) and oil globule diameter (0.165 ± 0.014 mm). The first division was observed approximately 45 min after fertilization. Blastula stage started after 4 h and the middle gastrula stage after 12:30 h. Early neurula was observed 17 h after fertilization. Cardiac beats and movements of the free embryonic tail were recorded after 30 h of incubation. Hatching occurred between 31 to 41 h and length of newly hatched larvae was 2.558 ± 0.051 mm. The embryonic development of this species is similar to previous studies regarding other marine fish. This study is the first report of embryonic development of A. scapularis, which is a valuable information that provide a baseline reference for the efforts for the culture of this species.

Characterization and Evolution of Dishevelled Genes in Paralichthys olivaceus

The study examined the key gene Dishevelled (Dvl or Dsh) in Wnt (Wingless and INT-1) signaling pathways. The gene (Dvl) was characterized in the flat fish Paralichthys olivaceus for its expression pattern structure and phylogenetics at the Ocean University of China in Qingdao, China in 2018. Three gene paralogues (Dvl1, Dvl2 and Dvl3) of the Dvl family were cloned in P. olivaceus and a N-terminal DAX domain, a central PDZ domain and a C-terminal DEP domain were discovered in all three protein paralogues. Phylogenetic analysis revealed that Dvl genes in P. olivaceus are most closely related to those in marine teleosts Larimichthys crocea and Stegastes partitus, followed by those in Cynoglossus semilaevis. For each Dvl gene, the genes in teleosts fall into a clade independent from the ones in other vertebrates, suggesting that the duplication of Dvl genes occurred prior to the divergence of vertebrates. The temporal expression patterns of the three Dvl genes were characterized during the embryonic development of teleosts. In P. olivaceus, all three Dvl genes remain at low expression levels during the early stages of development until gastrula stage, when the expression of Dvl1 was significantly up-regulated. The research revealed vastly different temporal expression patterns of Dvl genes and suggested that the structure of Dvl proteins is conserved, but the expression patterns of Dvl genes vary significantly among different classes.

Transcriptome analysis of regeneration during Xenopus laevis experimental twinning

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.