A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.

Cadmium affects retinogenesis during zebrafish embryonic development.

Ocular malformations are commonly observed in embryos of aquatic species after exposure to toxicants. Using zebrafish embryos as the model organism, we showed that cadmium exposure from sphere stage (4 hpf) to end of segmentation stage (24 hpf) induced microphthalmia in cadmium-treated embryos. Embryos with eye defects were then assessed for visual abilities. Cadmium-exposed embryos were behaviorally blind, showing hyperpigmentation and loss of camouflage response to light. We investigated the cellular basis of the formation of the small eyes phenotype and the induction of blindness by studying retina development and retinotectal projections. Retinal progenitors were found in cadmium-treated embryos albeit in smaller numbers. The number of retinal ganglion cells (RGC), the first class of retinal cells to differentiate during retinogenesis, was reduced, while photoreceptor cells, the last batch of retinal neurons to differentiate, were absent. Cadmium also affected the propagation of neurons in neurogenic waves. The neurons remained in the ventronasal area and failed to spread across the retina. Drastically reduced RGC axons and disrupted optic stalk showed that the optic nerves did not extend from the retina beyond the chiasm into the tectum. Our data suggested that impairment in neuronal differentiation of the retina, disruption in RGC axon formation and absence of cone photoreceptors were the causes of microphthalmia and visual impairment in cadmium-treated embryos.

Cadmium inhibits neurogenesis in zebrafish embryonic brain development.

Cadmium is a non-essential heavy metal found abundantly in the environment. Children of women exposed to cadmium during pregnancy display lower motor and perceptual abilities. High cadmium body burden in children is also related to impaired intelligence and lowered school achievement. However, little is known about the molecular and cellular basis of developmental neurotoxicity in the sensitive early life stages of animals. In this study, we explore neurological deficits caused by cadmium during early embryonic stages in zebrafish by examining regionalization of the neural tube, pattern formation and cell fate determination, commitment of proneural genes and induction of neurogenesis. We show that cadmium-treated embryos developed a smaller head with unclear boundaries between the brain subdivisions, particularly in the mid-hindbrain region. Embryos display normal anterior to posterior regionalization; however, the commitment of neural progenitor cells was affected by cadmium. We observe prominent reductions in the expression of several proneuronal genes including ngn1 in cell clusters, zash1a in the developing optic tectum, and zash1b in the telencephalon and tectum. Cadmium-treated embryos also have fewer differentiated neurons and glia in the facial sensory ganglia as indicated by decreased zn-12 expression. Also, a lower transcription level of neurogenic genes, ngn1 and neuroD, is observed in neurons. Our data suggest that cadmium-induced neurotoxicity can be caused by impaired neurogenesis, resulting in markedly reduced neuronal differentiation and axonogenesis.

Four-dimensional imaging and quantification of gene expression in early developing zebrafish (Danio rerio) embryos.

Four-dimensional (4D) imaging is a powerful tool for studying three-dimensional (3D) changes in an organism through time. Different imaging systems for obtaining 3D data from in vivo specimens have been developed but usually involved large and expensive machines. We successfully used a simple inverted compound microscope and a commercially available program to study and quantify in vivo changes in sonic hedgehog (shh) expression during early development in a green fluorescence protein (GFP) transgenic zebrafish (Danio rerio) line. We applied the 4D system to study the effect of 100 microM cadmium exposure on shh expression. In control zebrafish embryos, shh:GFP expression was detected at about 9 h post-fertilization (hpf) and increased steadily in the next 7 h, peaking at about 17 hpf and decreasing in the following 4 h. In the same time period, different shh expression volumes were observed in cadmium-treated and control embryos. Embryos affected by cadmium-exposure demonstrated a down-regulation in shh expression. The number of GFP-expressing cells measured by flow cytometry decreased, and expression of neurogenin-1, a downstream target of the shh signaling pathway, was down-regulated, providing additional supporting data on the effects of cadmium on shh. In summary, we demonstrated the setup of a 4D imaging system and its application to the quantification of gene expression.

C. elegans Stress-Induced Sleep Emerges from the Collective Action of Multiple Neuropeptides.

The genetic basis of sleep regulation remains poorly understood. In C. elegans, cellular stress induces sleep through epidermal growth factor (EGF)-dependent activation of the EGF receptor in the ALA neuron. The downstream mechanism by which this neuron promotes sleep is unknown. Single-cell RNA sequencing of ALA reveals that the most highly expressed, ALA-enriched genes encode neuropeptides. Here we have systematically investigated the four most highly enriched neuropeptides: flp-7, nlp-8, flp-24, and flp-13. When individually removed by null mutation, these peptides had little or no effect on stress-induced sleep. However, stress-induced sleep was abolished in nlp-8; flp-24; flp-13 triple-mutant animals, indicating that these neuropeptides work collectively in controlling stress-induced sleep. We tested the effect of overexpression of these neuropeptide genes on five behaviors modulated during sleep-pharyngeal pumping, defecation, locomotion, head movement, and avoidance response to an aversive stimulus-and we found that, if individually overexpressed, each of three neuropeptides (nlp-8, flp-24, or flp-13) induced a different suite of sleep-associated behaviors. These overexpression results raise the possibility that individual components of sleep might be specified by individual neuropeptides or combinations of neuropeptides.

Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis.

We have previously reported that exposure to cadmium during zebrafish embryonic development caused morphological malformations of organs and ectopic expression of genes involved in regulating developmental process. One of the most common developmental defects observed was altered axial curvature resulting from defects in the myotomes of the somites. In this study, we investigated the mechanisms of cadmium-induced toxicity in zebrafish somitogenesis. We showed that the critical period of exposure was the gastrulation period, which actually preceded the formation of the first morphologically distinct somites. The somites thus formed lost the typical chevron V-shape and are packed disorderly. The myogenic lineage commitment of the axial mesodermal cells was not affected, as the myogenic regulatory transcription factors were expressed normally. There were, however, losses of fast and slow muscle fibers in the myotomes. The innervation of the muscle blocks by spinal motoneurons is an important process of the somitogenesis. Both primary and secondary motoneurons appear to form normally while the axon growth is affected in cadmium-treated embryos. The notochord, which is essential in the patterning of the somites and the central nervous system, showed abnormal morphological features and failed to extend to the tail region. Taken together, it appears that cadmium exposure led to abnormal somite patterning of the muscle fibers and defects in axonogenesis.