Decoding an organ regeneration switch by dissecting cardiac regeneration enhancers.

Heart regeneration in regeneration-competent organisms can be accomplished through the remodeling of gene expression in response to cardiac injury. This dynamic transcriptional response relies on the activities of tissue regeneration enhancer elements (TREEs); however, the mechanisms underlying TREEs are poorly understood. We dissected a cardiac regeneration enhancer in zebrafish to elucidate the mechanisms governing spatiotemporal gene expression during heart regeneration. Cardiac lepb regeneration enhancer (cLEN) exhibits dynamic, regeneration-dependent activity in the heart. We found that multiple injury-activated regulatory elements are distributed throughout the enhancer region. This analysis also revealed that cardiac regeneration enhancers are not only activated by injury, but surprisingly, they are also actively repressed in the absence of injury. Our data identified a short (22 bp) DNA element containing a key repressive element. Comparative analysis across Danio species indicated that the repressive element is conserved in closely related species. The repression mechanism is not operational during embryogenesis and emerges when the heart begins to mature. Incorporating both activation and repression components into the mechanism of tissue regeneration constitutes a new paradigm that might be extrapolated to other regeneration scenarios.

An Accessible Protocol for the Generation of CRISPR-Cas9 Knockouts Using INDELs in Zebrafish.

The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.

Practical approaches for implementing forward genetic strategies in zebrafish.

The tropical fresh water minnow, Danio rerio, more commonly known as zebrafish, has emerged rapidly over the last decade as a powerful tool for developmental geneticists. External fertilization, high fecundity, a short generation time, and optical transparency of embryos during early development combined with the amenability to a variety of genetic manipulations constitute in the zebrafish the convergence of several unique advantages for a vertebrate model system. Traditional forward genetic screens, which employ the use of a chemical mutagen such as N-ethyl-N-nitrosourea to induce mutations in the male genome, have also proven to be highly successful in the zebrafish. This chapter provides experimental approaches to successfully induce pre-meiotic mutations in the male zebrafish germline and genetic strategies to recover and maintain such mutations in subsequent generations (Section 3.1). Though discussed specifically in the context of zebrafish research in this chapter, many of these genetic approaches may also be broadly applicable in other model systems. We also discuss experimental techniques to manipulate the ploidy of zebrafish embryos, which when used in combination with the standard mutagenesis protocol significantly expedite the identification of the induced mutations (Section 3.2). Additional stand-alone procedures are provided in Section 3.3, which are also required for the execution of the experiments discussed in its preceding sections.

Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4.

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.

The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote.

Embryos have been successfully used for the general study of the cell cycle. Although there are significant differences between the early embryonic and the somatic cell cycle in vertebrates, the existence of specialised factors that play a role during the early cell cycles has remained elusive. We analysed a lethal recessive maternal-effect mutant, futile cycle (fue), isolated in a maternal-effect screen for nuclear division defects in the zebrafish (Danio rerio). The pronuclei fail to congress in zygotes derived from homozygous fue mothers. In addition, a defect in the formation of chromosomal microtubules prevents mitotic spindle assembly and thus chromosome segregation in fue zygotes. However, centrosomal functions do not appear to be affected in fue embryos, suggesting this mutant blocks a subset of microtubule functions. Cleavage occurs normally for several divisions resulting in many anucleate cells, thus showing that nuclear- and cell division can be uncoupled genetically. Therefore, we propose that in mitotic spindle assembly chromosome-dependent microtubule nucleation is essential for the coupling of nuclear and cell division.