Isolation and molecular characterization of Rem2 isoforms in the rainbow trout (Oncorhynchus mykiss): Tissue and central nervous system expression

REM2 is a member of the REM, RAD, and GEM/KIR (RGK) subfamily of RAS superfamily proteins and plays an important role in brain development and function. In this study, two Rem2 isoforms were isolated from the rainbow trout (Oncorhynchus mykiss). The two genes, designated O. mykiss rem2a and rem2b, both encode 304 amino acid proteins with 61% and 62% identities to zebrafish (Danio rerio) Rem2, respectively, and each with 43% identity to mammalian (human) REM2. To our knowledge, this is the first incidence of Rem2 isoforms in a species that are the result of gene duplication. Both isoforms possessed similar tissue expression profiles with the highest levels in the brain. The rem2a gene has significantly higher expression levels than rem2b in all tissues assayed except the brain and head kidney. In the central nervous system, both isoforms showed similar expression levels with the highest levels occurring in the olfactory bulb, cerebrum, and midbrain, though rem2a expression is significantly higher in the spinal cord. Based on known functional roles of Rem2 in synapse development and stem cell proliferation, the characterization of Rem2 in rainbow trout could shed light on its role in adult vertebrate neurogenesis and brain regeneration.     

Transducin Duplicates in the Zebrafish Retina and Pineal Complex: Differential Specialisation after the Teleost Tetraploidisation

Gene duplications provide raw materials that can be selected for functional adaptations by evolutionary mechanisms. We describe here the results of 350 million years of evolution of three functionally related gene families: the alpha, beta and gamma subunits of transducins, the G protein involved in vision. Early vertebrate tetraploidisations resulted in separate transducin heterotrimers: gnat1/gnb1/gngt1 for rods, and gnat2/gnb3/gngt2 for cones. The teleost-specific tetraploidisation generated additional duplicates for gnb1, gnb3 and gngt2. We report here that the duplicates have undergone several types of subfunctionalisation or neofunctionalisation in the zebrafish. We have found that gnb1a and gnb1b are co-expressed at different levels in rods; gnb3a and gnb3b have undergone compartmentalisation restricting gnb3b to the dorsal and medial retina, however, gnb3a expression was detected only at very low levels in both larvae and adult retina; gngt2b expression is restricted to the dorsal and medial retina, whereas gngt2a is expressed ventrally. This dorsoventral distinction could be an adaptation to protect the lower part of the retina from intense light damage. The ontogenetic analysis shows earlier onset of expression in the pineal complex than in the retina, in accordance with its earlier maturation. Additionally, gnb1a but not gnb1b is expressed in the pineal complex, and gnb3b and gngt2b are transiently expressed in the pineal during ontogeny, thus showing partial temporal subfunctionalisation. These retina-pineal distinctions presumably reflect their distinct functional roles in vision and circadian rhythmicity. In summary, this study describes several functional differences between transducin gene duplicates resulting from the teleost-specific tetraploidisation.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish

In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 h post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.     

A new transgenic reporter line reveals expression of protocadherin 9 at a cellular level within the zebrafish central nervous system

The wiring of neuronal networks is far from understood. One outstanding question is how neurons of different types link up to form subnetworks within the greater context. Cadherins have been suggested to create an inclusion code where interconnected neurons express the same subtypes. Here, we have used a CRISPR/Cas9 knock-in approach to generate a transgenic zebrafish reporter line for protocadherin 9 (pcdh9), which is predominantly expressed within the central nervous system. Expression of eGFP was detected in subsets of neurons in the cerebellum, retina and spinal cord, in both larvae and juveniles. A closer characterization of the spinal locomotor network revealed that a portion of distinct classes of both excitatory and inhibitory interneurons, as well as motor neurons, expressed pcdh9. This transgenic line could thus be used to test the cadherin network hypothesis, through electrophysiological characterization of eGFP positive cells, to show if these are synaptically connected and form a discrete network within the spinal cord.     

Characterisation and differential expression during development of a duplicate Disabled-1 (Dab1) gene from zebrafish

We identified a new duplicated Dab1 gene (drDab1b) spanning around 25 kb of genomic DNA in zebrafish. Located in zebrafish chromosome 2, it is composed of 11 encoding exons and shows high sequence similarity to other Dab1 genes, including drDab1a, a zebrafish Dab1 gene previously characterised. drDab1b encodes by alternative splicing at least five different isoforms. Both drDab1a and drDab1b show differential gene expression levels in distinct adult tissues and during development. drDab1b is expressed in peripheral tissues (gills, heart, intestine, muscle), the immune system (blood, liver) and the central nervous system (CNS), whereas drDab1a is only expressed in gills, muscle and the CNS, suggesting a division of functions for two Dab1 genes in zebrafish adult tissues. RT-PCR analysis also reveals that both drDab1 genes show distinct developmental-specific expression patterns throughout development. drDab1b expression was higher than that of drDab1a, suggesting a major role of drDab1b in comparison with drDab1a during development and in different adult tissues. In addition, new putative Dab1 (a and/or b) from different teleost species were identified in silico and predicted protein products are compared with the previously characterised Dab1, demonstrating that the Dab1b group is more ancestral than their paralogue, the Dab1a group.     

Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system,gut and lateral line

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.     

Expression analysis of integrin β1 isoforms during zebrafish embryonic development

Integrins are a superfamily of the major metazoan receptors for cell–cell and cell–extracellular matrix adhesion. Integrins and their ligands play critical roles in a variety of fundamental cellular processes. Integrins are heterodimeric cell surface glycoproteins comprised of non-covalently bound α- and β-subunits. A variety of integrin subunits have been identified in mouse, chicken, zebrafish, Xenopus laevis and other vertebrates. In zebrafish multiple integrin β1 homologs have been identified. However, zebrafish embryo is a largely untapped model for analyzing integrin β1 isoforms temporal-spatial expression pattern, function and its relevance to human disease in whole animal level. Currently, we systematically analyzed the expression pattern of zebrafish integrin β1 including integrin beta 1a (itgb1a), integrin beta 1b (itgb1b), integrin beta 1b.1 (itgb1b.1), and integrin beta 1b.2 (itgb1b.2) at embryo stage using whole amount in situ hybridization. We show itgb1a, itgb1b and itgb1b.1 were maternally expressed in zygote, cleavage and blastula periods, while itgb1b.2 was not detectable in the corresponding stages. A more tissue specific pattern emerges during organogenesis, including heart expression for itgb1a, myotome borders for itgb1b, intestinal epithelium for itgb1b.1, and branchial arch for itgb1b.2. All are similarly expressed in the early embryonic epidermis and notochord. Additionally, itgb1a, itgb1b and itgb1b.2 shared the common expression in otic vesicle. Our study provides new insight into the integrin β1 expression and the use of this model organism to tackle future studies on the role of integrin β1 in embryo development.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1β rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.     

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.