Expression analysis of nel during zebrafish embryonic development

Nel is a multimeric extracellular glycoprotein which predominantly expressed in the nervous system and play an important role in neural development and functions. There are three nel paralogues included nell2a, nell2b, and nell3 in zebrafish, while systematic expression analysis of the nel family is still lacking. In this study, we performed a phylogenetic analysis on 7 species, in different species the nell2a are highly conserved, as is nell2b. Then, the expression profiles of nell2a, nell2b and nell3 were detected by in situ hybridization in zebrafish embryo, and the result showed that nel genes highly enriched in the central nervous system, but distributed in different regions of the brain. In addition, nell2a is also expressed in the olfactory pit, spinal cord, otic vesicle and retina (ganglion cell layer), nell2b was detected to express in gill arches, olfactory epithelium, olfactory pit, spinal cord, photoreceptor and retina (ganglion cell layer), it should be noted that the expression of nell3 is special, was only detected at 96 hpf in the brain and spinal cord of zebrafish. Overall, our results indicate that nell2a and nell2b genes are expressed in the nervous system and eyes of zebrafish embryo, while nell3 is expressed in different regions in the nervous system. The phylogenetic analysis also shows that nell3 sequences are significantly different from nell2a and nell2b. This study provides new evidence to better understand the role of nel in zebrafish embryo development.     

Expression analysis of the aquaporins during zebrafish embryonic development

The aquaporins are integral membrane proteins from a larger family of major intrinsic protein (MIP) that form pores in the membrane of cells. These proteins selectively transport water and other small uncharged solutes across cell plasma membranes. The organization of water within cells and tissues is fundamental to life, and the aquaporins play an important role in serving as the plumbing system for cells. As many as thirteen mammalian AQPs have been characterized, which have been shown to be vital for the regulation of water homeostasis in most tissues, such as renal water balance and brain-fluid homeostasis. However, complete expression patterns of most of the aquaporins in lower vertebrate at embryo stages has not been elucidated. Currently, we systematically described the temporal-spatial expression pattern of nine zebrafish aquaporins, using whole amount in situ hybridization. The results of whole mount in situ hybridization revealed that members of aquaporins family displayed diverse expression pattern, each of aquaporins has its unique distribution in different cell types and tissues, suggesting that they might play distinct roles in the embryonic development. Overall, current study will provide new insight into the expression of vertebrate quaporins and an important basis for the functional analysis of aquaporins in zebrafish development.     

Temporal and spatial expression of the four Igf ligands and two Igf type 1 receptors in zebrafish during early embryonic development

The insulin-like growth factor (Igf) family is an evolutionarily conserved system essential for normal growth and development in vertebrates. Unlike mammals, four distinct Igf ligands (Igf1, Igf2a, Igf2b and Igf3) and two Igf type 1 receptors (Igf1ra and Igf1rb) are present in zebrafish. However, the localization of these multiple ligands and receptors especially the recently discovered igf3 during early development of zebrafish is poorly understood. In this study, detailed expression patterns of these components of the Igf system during embryogenesis of zebrafish were analyzed. It was found that igf1 is specifically expressed in the trigeminal ganglia region from 18 hpf to 72 hpf, while igf2a is restricted to the caudal regions of the notochord from 14 hpf to 18 hpf as well as in the midbrain, dorsal hind brain and otic vesicle at 24 hpf. On the other hand, igf2a is highly expressed in the midbrain and pharyngeal arch region at 48 hpf, followed by its appearance in the liver and brain at 72 hpf, while igf2b is restricted to the floor plate and hypochord from 12 hpf to 18 hpf, and strong expression is also detected in the midbrain and dorsal hind brain at 24 hpf. The teleost specific igf3 is highly expressed in the pharyngeal arch region before 24 hpf, but is then restricted to the sternohyoideus after 48 hpf. The receptor subtype igf1ra is ubiquitously expressed before 24 hpf but is confined to the brain at 72 hpf. However, igf1rb is widely expressed before 10 hpf, but is more confined to the brain region at 24 hpf and 72 hpf. This dynamic temporal-spatial expression during embryogenesis of zebrafish, together with the unique and overlapping expression patterns of the Igf ligands and receptors suggest the coordination of the divergent functions of the Igf system during early development in zebrafish.     

Nonmuscle myosin II-B (myh10) expression analysis during zebrafish embryonic development

Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes (myh9, myh10, and myh14) encoding different nonmuscle myosin heavy chains. The three NM II isoforms share a very similar molecular structure and play important roles in a variety of fundamental biological processes. NM II-B (myh10) has been shown to be essential for the formation of mouse neural system and heart. But so far the complete knowledge for its expression in developing zebrafish embryos is lacking. In current study, we proved the conservation of zebrafish NM II-B in vertebrate evolution by in silicon analysis. Afterwards the NM II-B (myh10) expression was demonstrated to initiate after gastrulation stage. At 20 hpf, the expression is mainly restricted in central nervous system (CNS). It was maintained and expanded to sensor organ including eye, otic vesicle, and olfactory bulb at 36 hpf and later. We also detected myh10 mRNA hybridization signal in 48 hpf zebrafish heart. In addition, we investigated myh9a and myh9b mRNA distribution in zebrafish developing embryos. It was shown that myh10 and myh9 have distinct expression pattern, with myh9s not in neural system but in epidermis, enveloping layer (EVL). Our study provides new insight into the NM II expression and the use of this model organism to tackle future studies on the role of NM II in embryo development.     

Neurovascular development in the embryonic zebrafish hindbrain

The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos.     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

The embryonic expression patterns of zebrafish genes encoding LysM-domains

The function and structure of LysM-domain containing proteins are very diverse. Although some LysM domains are able to bind peptidoglycan or chitin type carbohydrates in bacteria, in fungi and in plants, the function(s) of vertebrate LysM domains and proteins remains largely unknown. In this study we have identified and annotated the six zebrafish genes of this family, which encode at least ten conceptual LysM-domain containing proteins. Two distinct sub-families called LysMD and OXR were identified and shown to be highly conserved across vertebrates. The detailed characterization of LysMD and OXR gene expression in zebrafish embryos showed that all the members of these sub-families are strongly expressed maternally and zygotically from the earliest stages of a vertebrate embryonic development. Moreover, the analysis of the spatio-temporal expression patterns, by whole mount and fluorescent in situ hybridizations, demonstrates pronounced LysMD and OXR gene expression in the zebrafish brain and nervous system during stages of larval development. None of the zebrafish LysMD or OXR genes was responsive to challenge with bacterial pathogens in embryo models of Salmonella and Mycobacterium infections. In addition, the expression patterns of the OXR genes were mapped in a zebrafish brain atlas.     

Distinct expression patterns of dickkopf genes during late embryonic development of Danio rerio

Dickkopf (dkk) genes belong to the family of secreted wnt-inhibitors with conserved cysteine-rich domains. In contrast to the prototype dkk1, dkk3 does not modulate canonical Wnt/β-catenin signalling. Until now, neither functions nor interaction partners of dkk3 in lower vertebrates have been described. In this study we cloned two dkk3 homologues dkk3a(dkk3l) and dkk3b(dkk3) and a dkk1 homologue dkk1a of the zebrafish and studied their expression patterns during embryonic development in comparison to the known dkk1b gene. Moreover, mutants with defects in hedgehog signalling (smo), notch (mib) signalling, nodal signalling (Zoep) or retinoic acid synthesis (neckless) were analyzed for changes in dkk3 gene expression.In situ hybridization analyses showed a dynamic expression of dkk1a and dkk1b primarily in epidermal structures of the otic vesicle, lens, branchial arches and fin folds. While dkk1a was expressed mainly in deep tissues, dkk1b expression was mainly found in protrusions at the outer surface of the branchial arch epidermis. In contrast, dkk3 genes showed expression in different tissues. Strong signals for dkk3a(dkk3l) were present in various neuronal structures of the head, whereas dkk3b(dkk3) expression was restricted mainly to endocrine cells of the pancreas and to the brachial arches.In summary, both dkk3 genes display a unique and distinct expression pattern in late embryonic development, pointing to a specific role during neuronal and pancreatic cell differentiation.     

Live imaging of endogenous Collapsin response mediator protein-1 expression at subcellular resolution during zebrafish nervous system development

Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are functionally important during vertebrate development. We have generated a zebrafish gene trap line that produces fluorescently tagged Crmp1 protein, which can be dynamically tracked in living fish at subcellular resolution. The results show that Crmp1 is expressed in numerous sites in the developing nervous system. Early expression is apparent in the forebrain, epiphysis, optic tectum and the developing spinal cord. In the larval brain, Crmp1 is expressed in several distinct brain regions, such as the telencephalon, habenula and cerebellum. In addition, it is expressed in the spinal cord in a manner that persists in the larva. The results suggest that this Crmp1 protein trap line offers a powerful tool to track selected neuronal populations at high resolution.     

Differential expression of two somatostatin genes during zebrafish embryonic development

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.     

The zebrafish Znt1asa17 mutant reveals roles of zinc transporter-1a in embryonic development

BackgroundZinc is one of the vital micronutrients required through various developmental stages in animals. Zinc transporter-1 (ZnT1; Slc30a1) is essential in vertebrates for nutritional zinc uptake and cellular zinc extrusion. Knockout of ZnT1 is lethal in vertebrates and there are therefore few functional studies of this protein in vivo.MethodsIn the present study we characterised the embryonic development in a zebrafish Znt1a mutant (Znt1a^sa17) which is lacking the last 40 amino acids of Znt1a as generated by TILLING. In parallel experiments, we compared the development of a zebrafish embryo Znt1a morphant (Znt1a^MO) which was generated by knockdown of Znt1a using morpholino-modified oligonucliotides.ResultsThe homozygous Znt1a^sa17 embryo is viable, but displays a subtle phenotype informing on the biological roles of Znt1a. The Znt1a^sa17 fish have delayed development, including attenuated epiboly. They further show a decrease in phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2), retarded yolk resorption, and impaired clearance of free Zn²⁺ from the vitelline fluid and its storage in hatching gland cells. All these aberrations are milder versions of those observed upon knockdown of Znt1a by morpholinos. Interestingly, the phenotype could be rescued by addition of the cell-permeable zinc chelator, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) to the incubation medium and was aggravated by addition of zinc(II). Thus, the Znt1a^sa17 mutant has a reduced ability to handle zinc and can be characterised as a hypomorph.ConclusionThis study is the first to show that the last 40 amino acids of Znt1a are of importance for its role in zinc homeostasis and ability to activate the MAPK/ERK pathway contrary to what was previously thought.