No description available.

The development of swimming was investigated in zebrafish aged 1.5 to 5 days postfertilization by examining both the swimming behavior and its generation by the nervous system. Upon hatching (at day 2), swimming is undirected and occurs in sustained bursts of high frequency (mean = 67 Hz) tail undulations. By 4 days, the swimming pattern matures to a more directed, less erratic, beat-and-glide pattern where slower (mean = 35 Hz) tail undulations, lasting ∼200 ms, alternate with longer gliding rest periods. Swimming is powered by two classes of embryonic muscles (embryonic red, ER and white, EW) that are electrically coupled within (but not between) classes and have physiological properties similar to vertebrate tonic and twitch muscle, respectively. ER fibers have a lower chloride ion permeability than EW fibers and do not have sodium dependent action potentials. In paralyzed preparations, motoneurons and muscle fibers received coordinated excitatory synaptic activity (with left to right alternation and head to tail propagation) corresponding to either burst or beat-and-glide swimming. ER muscle was de-recruited at the fastest swimming rates and EW fibers dropped out at the slowest swimming rates. Rhythmic motoneuron output was generated by a phasic glutamatergic and a largely tonic glycinergic synaptic drive. Glutamatergic synapses had either or both AMPA/kainate and NMDA receptors and the kinetics of these synaptic currents were fixed throughout the developmental period examined. When depolarized, motoneurons fired high frequency (up to 800 Hz) bursts of action potentials that rapidly accommodated (within ∼20 ms) due to voltage and calcium dependent outwardly rectifying conductances. These intrinsic motoneuron properties are hypothesized to interact with the rhythmic synaptic drive to pattern motor output (at ∼25--75 Hz) to locomotor muscles. The neural generation of swimming in developing zebrafish is thus fundamentally similar to locomotion in adu

In embryonic neurons, the intracellular chloride concentration is elevated, making GABA and glycine depolarizing. Later in development, coincident with neuronal maturation, the extruding potassium-chloride co-transporter KCC2 is expressed. It reverses the chloride gradient, rendering it hyperpolarizing. Early depolarization is assumed to play trophic roles during nervous system development. I thus decided to investigate the effects of the depolarizing chloride gradient on development in vivo in the zebrafish embryo. I first determined the temporal pattern of KCC2 expression in zebrafish and found it was absent in the embryo. I then over-expressed wild-type, gain-of-function and loss-of-function variants of human KCC2, using GFP-tagged constructs for detection purposes. Over-expression of functional hKCC2 perturbed the morphology and motor behaviours of the embryos. At the cellular level, KCC2 impaired axonal growth and affected the neuronal populations in the brain, hindbrain and spinal cord. This suggests the depolarizing effects of glycine are critical for neurogenesis.

No description available.

Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.

microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 5, 2012 - Dec. 5, 2013