116 research outputs found
FGF Signaling Mediates Regeneration of the Differentiating Cerebellum through Repatterning of the Anterior Hindbrain and Reinitiation of Neuronal Migration
To address the regenerative capability of the differentiating hindbrain, we ablated the cerebellum in wild-type and transgenic zebrafish embryos. These larvae showed no obvious locomotive malfunction several days after the ablation. Expression analysis and in vivo time-lapse recording in GFP (green fluorescent protein)-transgenic embryos indicate that cerebellar neuronal cells can regenerate from the remaining anterior hindbrain. The onset of regeneration is accompanied by repatterning within the anterior hindbrain. Inhibition of FGF signaling immediately after cerebellar ablation results in the lack of regenerating cerebellar neuronal cells and the absence of cerebellar structures several days later. Moreover, impaired FGF signaling inhibits the repatterning of the anterior hindbrain and the reexpression of rhombic lip marker genes soon after cerebellar ablation. This demonstrates that the hindbrain is highly plastic in recapitulating early embryonic differentiation mechanisms during regeneration. Moreover, the regenerating system offers a means to uncouple cerebellar differentiation from complex morphogenetic tissue rearrangements
Direct imaging of in vivo neuronal migration in the developing cerebellum
The upper rhombic lip (URL), a germinal zone in the dorsoanterior hindbrain, has long been known to be a source for neurons of the vertebrate cerebellum [1]. It was thought to give rise to dorsally migrating granule cell precursors [2](Figure 1e); however, recent fate mapping studies have questioned the exclusive contributions of the URL to granule cells 3, 4 and 5. By taking advantage of the clarity of the zebrafish embryo during the stages of brain morphogenesis, we have followed the fate of neuronal precursor cells generated within the upper rhombic lip directly. Combining a novel GFP labeling strategy with in vivo time-lapse imaging, we find, contrary to the former view, that most URL-descendants migrate anterior toward the midhindbrain boundary (MHB) and then course ventrally along the MHB (Figure 1f). As the migrating neuronal precursors reach the MHB, they form ventrally extending projections, likely axons, and continue ventral migration to settle outside of the cerebellum, in the region of the ventral brainstem. Thus, we define a new pathway for URL-derived neuronal precursor cells consistent with the recent fate maps. In addition, our results strongly suggest that the MHB plays a crucial role, not only in induction and patterning of the cerebellar anlage, but also in organizing its later morphogenesis by influencing cell migration
The zebrafish cerebellar rhombic lip is spatially patterned in producing granule cell populations of different functional compartments
AbstractThe upper rhombic lip, a prominent germinal zone of the cerebellum, was recently demonstrated to generate different neuronal cell types over time from spatial subdomains. We have characterized the differentiation of the upper rhombic lip derived granule cell population in stable GFP-transgenic zebrafish in the context of zebrafish cerebellar morphogenesis. Time-lapse analysis followed by individual granule cell tracing demonstrates that the zebrafish upper rhombic lip is spatially patterned along its mediolateral axis producing different granule cell populations simultaneously. Time-lapse recordings of parallel fiber projections and retrograde labeling reveal that spatial patterning within the rhombic lip corresponds to granule cells of two different functional compartments of the mature cerebellum: the eminentia granularis and the corpus cerebelli. These cerebellar compartments in teleosts correspond to the mammalian vestibulocerebellar and non-vestibulocerebellar system serving balance and locomotion control, respectively. Given the high conservation of cerebellar development in vertebrates, spatial partitioning of the mammalian granule cell population and their corresponding earlier-produced deep nuclei by patterning within the rhombic lip may also delineate distinct functional compartments of the cerebellum. Thus, our findings offer an explanation for how specific functional cerebellar circuitries are laid down by spatio-temporal patterning of cerebellar germinal zones during early brain development
All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish
The zebrafish Danio rerio is a valuable and common model for scientists in the fields of genetics and developmental biology. Since zebrafish are also amenable to genetic manipulation, modelling of human diseases or behavioral experiments have moved into the focus of zebrafish research. Consequently, gene expression data beyond embryonic and larval stages become more important, yet there is a dramatic knowledge gap of gene expression beyond day four of development. Like in other model organisms, the visualization of spatial and temporal gene expression by whole mount in situ hybridization (ISH) becomes increasingly difficult when zebrafish embryos develop further and hence the growing tissues become dense and less permeable. Here we introduce a modified method for whole mount ISH, which overcomes these penetration and detection problem. The method is an all in one solution that enables the detection and visualization of gene expression patterns up to the late larval stage in a 3D manner without the need for tissue sectioning and offers a valuable extension for whole mount ISH by immunohistochemistry in the zebrafish field
Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines
In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis
NeuroExaminer: an all-glass microfluidic device for whole-brain in vivo imaging in zebrafish
While microfluidics enables chemical stimuli application with high spatio-temporal precision, light-sheet microscopy allows rapid imaging of entire zebrafish brains with cellular resolution. Both techniques, however, have not been combined to monitor whole-brain neural activity yet. Unlike conventional microfluidics, we report here an all-glass device (NeuroExaminer) that is compatible with whole-brain in vivo imaging using light-sheet microscopy and can thus provide insights into brain function in health and disease
Die Wattsedimente in der nördlichen und mittleren Meldorfer Bucht Untersuchungen zu Fragen der Sedimentverteilung und der Schlicksedimentation
The Long Adventurous Journey of Rhombic Lip Cells in Jawed Vertebrates: A Comparative Developmental Analysis
This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishe
Quantitative mapping of intracardiac blood flow in embryonic zebrafish
Using real-time in vivo imaging and digital particle image velocimetry (DPIV) we quantitatively described the intracardiac flow environment of early zebrafish (Danio rerio) embryos. Gross cardiac dynamics were defined for two embryonic stages: 4.5 days post fertilization (dpf) and 37 hours post fertilization (hpf) using high-speed transmitted light microscopy with valve dynamics visualized through high-speed laser-scanning microscopy on transgenic embryos expressing GFP
S-Sulfocysteine Induces Seizure-Like Behaviors in Zebrafish
Sulfite is a neurotoxin, which is detoxified by the molybdenum cofactor (Moco)-dependent enzyme sulfite oxidase (SOX). In humans, SOX deficiency causes the formation of the glutamate analog S-Sulfocysteine (SSC) resulting in a constant overstimulation of ionotropic glutamatergic receptors. Overstimulation leads to seizures, severe brain damage, and early childhood death. SOX deficiency may be caused either by a mutated sox gene or by mutations in one of the genes of the multi-step Moco biosynthesis pathway. While patients affected in the first step of Moco biosynthesis can be treated by a substitution therapy, no therapy is available for patients affected either in the second or third step of Moco biosynthesis or with isolated SOX deficiency. In the present study, we used a combination of behavior analysis and vital dye staining to show that SSC induces increased swimming, seizure-like movements, and increased cell death in the central nervous system of zebrafish larvae. Seizure-like movements were fully revertible upon removal of SSC or could be alleviated by a glutamatergic receptor antagonist. We conclude that in zebrafish SSC can chemically induce phenotypic characteristics comparable to the disease condition of human patients lacking SOX activity
- …