Germline-specific dgcr8 knockout in zebrafish using a BACK approach

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.     

Micromanaging freeze tolerance: the biogenesis and regulation of neuroprotective microRNAs in frozen brains

When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up to?~?65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze–thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica. Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.     

Role of p21-activated kinases in cardiovascular development and function

p21-activated kinases (Paks) are a group of six serine/threonine kinases (Pak1-6) that are involved in a variety of biological processes. Recently, Paks, more specifically Pak1, -2, and -4, have been shown to play important roles in cardiovascular development and function in a range of model organisms including zebrafish and mice. These functions include proper morphogenesis and conductance of the heart, cardiac contractility, and development and integrity of the vasculature. The mechanisms underlying these effects are not fully known, but they likely differ among the various Pak isoforms and include both kinase-dependent and -independent functions. In this review, we discuss aspects of Pak function relevant to cardiovascular biology as well as potential therapeutic implications of small-molecule Pak inhibitors in cardiovascular disease.     

Heat shock protein gene expression during embryonic development of the zebrafish

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.     

The Akt-associated microRNAs

As master gene regulators, microRNAs are involved in diverse cellular pathways. It is well known that microRNAs are often dysregulated in many types of cancer and other human diseases. In cancer, microRNAs may function as oncogenes or tumor suppressors. Interestingly, recent evidence suggests that microRNA-mediated gene regulation interconnects with the Akt pathway, forming an Akt–microRNA regulatory network. MicroRNAs and Akt in this network work together to exert their cellular functions. Thus, a better understanding of this Akt–microRNA regulatory network is critical to successful targeting of the PI3K/Akt pathway for cancer therapy. We review recent advances in the understanding of how microRNAs affect Akt activity as well as how microRNAs are regulated through the Akt pathway. We also briefly discuss the clinical implication of gene regulation mediated through Akt-associated microRNAs.     

The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis

The zinc finger of the cerebellum gene (ZIC) discovered in Drosophila melanogaster (odd-paired) has five homologs in Xenopus, chicken, mice, and humans, and seven in zebrafish. This pattern of gene copy expansion is accompanied by a divergence in gene and protein structure, suggesting that Zic family members share some, but not all, functions. ZIC genes are implicated in neuroectodermal development and neural crest cell induction. All share conserved regions encoding zinc finger domains, however their heterogeneity and specification remain unexplained. In this review, the evolution, structure, and expression patterns of the ZIC homologs are described; specific functions attributable to individual family members are supported. A review of data from functional studies in Xenopus and murine models suggest that ZIC genes encode multifunctional proteins operating in a context-specific manner to drive critical events during embryogenesis. The identification of ZIC mutations in congenital syndromes highlights the relevance of these genes in human development.     

Genetics of photoreceptor degeneration and regeneration in zebrafish

Zebrafish are unique in that they provide a useful model system for studying two critically important problems in retinal neurobiology, the mechanisms responsible for triggering photoreceptor cell death and the innate stem cell–mediated regenerative response elicited by this death. In this review we highlight recent seminal findings in these two fields. We first focus on zebrafish as a model for studying photoreceptor degeneration. We summarize the genes currently known to cause photoreceptor degeneration, and we describe the phenotype of a few zebrafish mutants in detail, highlighting the usefulness of this model for studying this process. In the second section, we discuss the several different experimental paradigms that are available to study regeneration in the teleost retina. A model outlining the sequence of gene expression starting from the dedifferentiation of Müller glia to the formation of rod and cone precursors is presented.     

Tumor surveillance by circulating microRNAs: a hypothesis

A growing body of experimental evidence supports the diagnostic relevance of circulating microRNAs in various diseases including cancer. The biological relevance of circulating microRNAs is, however, largely unknown, particularly in healthy individuals. Here, we propose a hypothesis based on the relative abundance of microRNAs with predominant tumor suppressor activity in the blood of healthy individuals. According to our hypothesis, certain sets of circulating microRNAs might function as a tumor surveillance mechanism exerting continuous inhibition on tumor formation. The microRNA-mediated tumor surveillance might complement cancer immune surveillance.     

Chromosomal organization at the level of gene complexes

Metazoan genomes primarily consist of non-coding DNA in comparison to coding regions. Non-coding fraction of the genome contains cis-regulatory elements, which ensure that the genetic code is read properly at the right time and space during development. Regulatory elements and their target genes define functional landscapes within the genome, and some developmentally important genes evolve by keeping the genes involved in specification of common organs/tissues in clusters and are termed gene complex. The clustering of genes involved in a common function may help in robust spatio-temporal gene expression. Gene complexes are often found to be evolutionarily conserved, and the classic example is the hox complex. The evolutionary constraints seen among gene complexes provide an ideal model system to understand cis and trans-regulation of gene function. This review will discuss the various characteristics of gene regulatory modules found within gene complexes and how they can be characterized.     

Experimental verification of a conserved intronic microRNA located in the human <Emphasis Type="Italic">TrkC</Emphasis> gene with a cell type-dependent apoptotic function

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75^NTR receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.     

Cardiomyocyte proliferation in zebrafish and mammals: lessons for human disease

Cardiomyocytes proliferate profusely during early development and for a brief period after birth in mammals. Within a month after birth, this proliferative capability is dramatically reduced in mammals unlike lower vertebrates where it persists into adult life. The zebrafish, for example, retains the ability to regenerate the apex of the heart following resection by a mechanism predominantly driven by cardiomyocyte proliferation. Differences in proliferative capacity of cardiomyocytes in adulthood between mammals and lower vertebrates are closely liked to ontogenetic or phylogenetic factors. Elucidation of these factors has the potential to provide enormous benefits if they lead to the development of therapeutic strategies that facilitate cardiomyocyte proliferation. In this review, we highlight the differences between Mammalian and Zebrafish cardiomyocytes, which could explain at least in part the different proliferative capacities in these two species. We discuss the advantages of the zebrafish as a model of cardiomyocyte proliferation, particularly at the embryonic stage. We also identify a number of key molecular pathways with potential to reveal key steps in switching cardiomyocytes from a quiescent to a proliferative phenotype.