Nick R. Love

Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration

BackgroundThe molecular mechanisms governing vertebrate appendage regeneration remain poorly understood. Uncovering these mechanisms may lead to novel therapies aimed at alleviating human disfigurement and visible loss of function following injury. Here, we explore tadpole tail regeneration in Xenopus tropicalis, a diploid frog with a sequenced genome.ResultsWe found that, like the traditionally used Xenopus laevis, the Xenopus tropicalis tadpole has the capacity to regenerate its tail following amputation, including its spinal cord, muscle, and major blood vessels. We examined gene expression using the Xenopus tropicalis Affymetrix genome array during three phases of regeneration, uncovering more than 1,000 genes that are significantly modulated during tail regeneration. Target validation, using RT-qPCR followed by gene ontology (GO) analysis, revealed a dynamic regulation of genes involved in the inflammatory response, intracellular metabolism, and energy regulation. Meta-analyses of the array data and validation by RT-qPCR and in situ hybridization uncovered a subset of genes upregulated during the early and intermediate phases of regeneration that are involved in the generation of NADP/H, suggesting that these pathways may be important for proper tail regeneration.ConclusionsThe Xenopus tropicalis tadpole is a powerful model to elucidate the genetic mechanisms of vertebrate appendage regeneration. We have produced a novel and substantial microarray data set examining gene expression during vertebrate appendage regeneration.

pTransgenesis:a cross-species, modular transgenesis resource

As studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.

C/EBPα initiates primitive myelopoiesis in pluripotent embryonic cells

The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Loss- and gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally, we use live imaging to show that cebpa-expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus, C/EPBalpha is the first known single factor capable of initiating an entire myelopoiesis pathway in pluripotent cells in the embryo.

Carbohydrate metabolism during vertebrate appendage regeneration: What is its role? How is it regulated? A postulation that regenerating vertebrate appendages facilitate glycolytic and pentose phosphate pathways to fuel macromolecule biosynthesis

We recently examined gene expression during Xenopus tadpole tail appendage regeneration and found that carbohydrate regulatory genes were dramatically altered during the regeneration process. In this essay, we speculate that these changes in gene expression play an essential role during regeneration by stimulating the anabolic pathways required for the reconstruction of a new appendage. We hypothesize that during regeneration, cells use leptin, slc2a3, proinsulin, g6pd, hif1α expression, receptor tyrosine kinase (RTK) signaling, and the production of reactive oxygen species (ROS) to promote glucose entry into glycolysis and the pentose phosphate pathway (PPP), thus stimulating macromolecular biosynthesis. We suggest that this metabolic shift is integral to the appendage regeneration program and that the Xenopus model is a powerful experimental system to further explore this phenomenon.

A simple method of transgenesis using I-sce I meganuclease in Xenopus.

Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for generating transgenic lines in Xenopus laevis and Xenopus tropicalis. In addition to it being simpler than the REMI method, this protocol also results in single copy integration events rather than tandem concatemers. Although the protocol we describe is for X. tropicalis, the method can also be used to generate transgenic lines in X. laevis. We also describe a convenient method for designing and generating complex constructs for transgenesis, named pTransgenesis, based on the Multisite Gateway(®) cloning, which include I-SceI sites and Tol2 elements to facilitate genome integration.

NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms

Significance Metabolism relies on a set of molecules that provide the chemical framework for all cellular activities. Among these molecules is NADP, a metabolite synthesized from vitamin B3 that is critical for basic metabolism, calcium signaling, and antiinflammatory processes. Despite NADP’s fundamental importance, very little is known about how animal cells regulate their NADP pool. This study shows that the enzyme NAD kinase is required for maintaining NADP levels in animals, is essential for embryonic development, and exhibits conserved regulatory mechanisms among evolutionarily diverse animals such as humans and sea urchins. Together, these results reveal new insights into why vitamin B3 is essential and how it is converted to NADP, and suggests new therapeutic avenues to improve human and animal metabolism. Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD+ kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca2+/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

Tadpole tail regeneration in Xenopus

Some organisms have a remarkable ability to heal wounds without scars and to regenerate complex tissues following injury. By gaining a more complete understanding of the biological mechanisms that promote scar-free healing and tissue regeneration, it is hoped that novel treatments that can enhance the healing and regenerative capacity of human patients can be found. In the present article, we briefly examine the genetic, molecular and cellular mechanisms underlying the regeneration of the Xenopus tadpole tail.

Comparative, transcriptome analysis of self-organizing optic tissues

Embryonic stem (ES) cells have a remarkable capacity to self-organize complex, multi-layered optic cups in vitro via a culture technique called SFEBq. During both SFEBq and in vivo optic cup development, Rax (Rx) expressing neural retina epithelial (NRE) tissues utilize Fgf and Wnt/β-catenin signalling pathways to differentiate into neural retina (NR) and retinal-pigmented epithelial (RPE) tissues, respectively. How these signaling pathways affect gene expression during optic tissue formation has remained largely unknown, especially at the transcriptome scale. Here, we address this question using RNA-Seq. We generated Rx+ optic tissue using SFEBq, exposed these tissues to either Fgf or Wnt/β-catenin stimulation, and assayed their gene expression across multiple time points using RNA-Seq. This comparative dataset will help elucidate how Fgf and Wnt/β-catenin signaling affect gene expression during optic tissue differentiation and will help inform future efforts to optimize in vitro optic tissue culture technology.

Ca2+-Induced Mitochondrial ROS Regulate the Early Embryonic Cell Cycle

Summary While it is appreciated that reactive oxygen species (ROS) can act as second messengers in both homeostastic and stress response signaling pathways, potential roles for ROS during early vertebrate development have remained largely unexplored. Here, we show that fertilization in Xenopus embryos triggers a rapid increase in ROS levels, which oscillate with each cell division. Furthermore, we show that the fertilization-induced Ca2+ wave is necessary and sufficient to induce ROS production in activated or fertilized eggs. Using chemical inhibitors, we identified mitochondria as the major source of fertilization-induced ROS production. Inhibition of mitochondrial ROS production in early embryos results in cell-cycle arrest, in part, via ROS-dependent regulation of Cdc25C activity. This study reveals a role for oscillating ROS levels in early cell cycle regulation in Xenopus embryos.

Carbohydrate metabolism during vertebrate appendage regeneration: What is its role? How is it regulated?

We recently examined gene expression during Xenopus tadpole tail appendage regeneration and found that carbohydrate regulatory genes were dramatically altered during the regeneration process. In this essay, we speculate that these changes in gene expression play an essential role during regeneration by stimulating the anabolic pathways required for the reconstruction of a new appendage. We hypothesize that during regeneration, cells use leptin, slc2a3, proinsulin, g6pd, hif1α expression, receptor tyrosine kinase (RTK) signaling, and the production of reactive oxygen species (ROS) to promote glucose entry into glycolysis and the pentose phosphate pathway (PPP), thus stimulating macromolecular biosynthesis. We suggest that this metabolic shift is integral to the appendage regeneration program and that the Xenopus model is a powerful experimental system to further explore this phenomenon.