Zebrafish (Danio rerio) serve as a very useful model organism because they have a fast generation time, clear embryos and a well mapped genome. In this research project, the students in the Developmental Biology lab course and the students in the Freshmen Research Initiative have used these characteristics to conduct a screening of the zebrafish genome in order to identify genes that are required for development. The CRISPR/Cas9 system (a protein that creates double strand breaks at specific sites in the genome that are then repaired by the cellular machinery) was recently specialized for the use in zebrafish. However, there are usually mistakes made when repairing the break. By using this system we can create mutations at specific sites in the genome and even delete entire sections. We can then observe if the mutation has created any notable phenotypes in the developing embryo. That information can give us insight into what the genetic requirements are for development or how those mechanisms can go wrong in diseases such as cancer.

Objective—The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis.

Approach and Results—Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites.

Conclusions—Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.

As a longtime BSA member (64 years), I have attended all but two BSA annual meetings and have literally rubbed shoulders with some of the best and most famous botanists in the world (in audience at my first talk as a young graduate student were Ledyard Stebbins, Katherine Esau, Ernest Gifford and Vernon Cheadle)! How fortunate (and scared) can one be! And now as a long-time member of BSA, I reflect on how I have committed part of my professional outside-of-the-university time in a variety of ways to the Society—they include financial support, oral and poster presentations, publications in AJB, section chair, committee member, Board of Directors and Council member, Treasurer, and yes, President. All of these involvements have been extremely rewarding to me both professionally and personally.

Plants synthesize a huge variety of terpenoid natural products, including photosynthetic pigments, signaling molecules and defensive substances. These are often produced as complex mixtures, presumably shaped by selective pressure over evolutionary timescales, some of which have been found to have pharmaceutical and other industrial uses. Elucidation of the relevant biosynthetic pathways can provide increased access (e.g., via molecular breeding or metabolic engineering), and enable reverse genetic approaches towards understanding the physiological role of these natural products in plants as well. While such information can be obtained via a variety of approaches, this review describes the emerging use of synthetic biology to recombinantly reconstitute plant terpenoid biosynthetic pathways in heterologous host organisms as a functional discovery tool, with a particular focus on incorporation of the historically problematic cytochrome P450 mono-oxygenases. Also falling under the synthetic biology rubric and discussed here is the nascent application of genome-editing tools to probe physiological function.

Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize g lossy2 ( gl2 ) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

Super-resolution fluorescence microscopy has generated tremendous success in revealing detailed subcellular structures in animal cells. However, its application to plant cell biology remains extremely limited due to numerous technical challenges, including the generally high fluorescence background of plant cells and the presence of the cell wall. In the current study, stochastic optical reconstruction microscopy (STORM) imaging of intact Arabidopsis thaliana seedling roots with a spatial resolution of 20–40 nm was demonstrated. Using the super-resolution images, the spatial organization of cortical microtubules in different parts of a whole Arabidopsis root tip was analyzed quantitatively, and the results show the dramatic differences in the density and spatial organization of cortical microtubules in cells of different differentiation stages or types. The method developed can be applied to plant cell biological processes, including imaging of additional elements of the cytoskeleton, organelle substructure, and membrane domains.

A study of yolks stored up to 168 d at −20 °C was conducted to determine the gelation behavior and mechanism of freeze–thawed yolk. Methods used were rheology, native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (native- and SDS-PAGE), differential scanning calorimetry (DSC), transmission electron microscopy (TEM), particle size analysis, and proton nuclear magnetic resonance (1H NMR) spectroscopy for matrix mobility. Results indicate that both constituents of plasma and granules contributed to gelation of yolk under freezing. PAGE analyses suggest that granular proteins participated in aggregation during freeze–thaw. Increasing gel strength and particle size and decreasing water and lipid–water mobility indicate that lipoproteins or apolipoproteins aggregated. At storage times ≥84 d, increased protein and lipid mobility, the detection of smaller particles, and secondarily increased gel strength suggest the liberation of protein or lipoprotein components from previously formed aggregates and further aggregation of these constituents. Disruption of the gelled yolk matrix observed with TEM supported that ice crystal formation was required for gelation to occur. A two-stage dynamic gelation model is thus proposed.

The enteric nervous system (ENS) consists of neurons and glia that control motility, secretions, and blood flow within the gastrointestinal tract. Using the zebrafish, Danio rerio, as a model we aim to understand ENS development and how this process might go array in disorders such as Hirschsprung’s, in which children are born lacking neurons in portions of their gastrointestinal tract. The number of markers for enteric glia is relatively small and many of the current immunohistochemical approaches are confounded by the uncertainty of cross-reactivity patterns between species. In preliminary experiments using established glia markers, we obtained unexpected results identifying glia in mutants lacking the ENS, suggesting a lack of glia marker specificity, or the presence of an uncharacterized subpopulation of glia in our mutants. To distinguish between these possibilities, we have cloned other markers to examine glia populations in normal larvae and larvae with defects in ENS development.

In this article, we further explore the data generated for the research article “Onecut1 and Onecut2 play critical roles in the development of the mouse retina”. To better understand the functionality of the Onecut family of transcription factors in retinogenesis, we investigated the retinal transcriptomes of developing and mature mice to identify genes with differential expression. This data article reports the full transcriptomes resulting from these experiments and provides tables detailing the differentially expressed genes between wildtype and Onecut1 or 2 deficient retinas. The raw array data of our transcriptomes as generated using Affymetrix microarrays are available on the NCBI Gene Expression Omnibus (GEO) browser (Reference number GSE57917 and GSE57918).

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. Once these nerve cells die, the patient’s muscles waste away, resulting in paralysis and eventually death. Two primary forms of ALS exist: Familial and Sporadic. Familial constitutes 5-10% of cases and is defined by ALS being present in one or more cases in a family’s lineage. Sporadic makes up 90-95% of ALS cases and is essentially when no family history exists with ALS but an individual has ALS. Mutations in SOD1 have been the most studied in regards to ALS. However there are many other genes linked to ALS that have not been studied. VCP is a gene that has been linked to several different diseases including familial versions of ALS. The protein has been linked to many different cellular processes including protein degradation and programmed cell death. To gain a better understanding into the development and eventual death of motor neurons, we are using both TAL-effector nuclease (TALEN) mediated mutagenesis and a VCP CRSPR to create zebrafish that are mutant for VCP. These mutant fish will hopefully allow us to create a new model of motor neuron degeneration or ALS.