Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse betaglobin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein Cerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

Overview of mathematical approaches used to model bacterial chemotaxis II: bacterial populations

We review the application of mathematical modeling to understanding the behavior of populations of chemotactic bacteria. The application of continuum mathematical models, in particular generalized Keller–Segel models, is discussed along with attempts to incorporate the microscale (individual) behavior on the macroscale, modeling the interaction between different species of bacteria, the interaction of bacteria with their environment, and methods used to obtain experimentally verified parameter values. We allude briefly to the role of modeling pattern formation in understanding collective behavior within bacterial populations. Various aspects of each model are discussed and areas for possible future research are postulated

The AMT1 Arginine Methyltransferase Gene Is Important for Plant Infection and Normal Hyphal Growth in Fusarium graminearum

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling.

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants

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Kelly Sheehan-Rooney is with UT Austin, Mary E. Swartz is with UT Austin, C. Ben Lovely is with UT Austin, Michael J. Dixon is with University of Manchester, Johann K. Eberhart is with UT Austin.In human, mutation of the transcription factor SATB2 causes severe defects to the palate and jaw. The expression and sequence of SATB2 is highly conserved across vertebrate species, including zebrafish. We sought to understand the regulation of satb2 using the zebrafish model system. Due to the normal expression domains of satb2, we analyzed satb2 expression in mutants with disrupted Hh signaling or defective ventral patterning. While satb2 expression appears independent of Edn1 signaling, appropriate expression requires Shha, Smo, Smad5 and Hand2 function. Transplantation experiments show that neural crest cells receive both Bmp and Hh signaling to induce satb2 expression. Dorsomorphin- and cyclopamine-mediated inhibition of Bmp and Hh signaling, respectively, suggests that proper satb2 expression requires a relatively earlier Bmp signal and a later Hh signal. We propose that Bmp signaling establishes competence for the neural crest to respond to Hh signaling, thus inducing satb2 expression.This work was supported by United States National Institutes of Health grants DE018088 and DE020884 to J.K.E. and MRC grant (G1001601) and support from the Healing Foundation to M.J.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Endodermal signals are required for <i>satb2</i> expression in ventral arches 2–7.

<p>(A) At 38 hpf, <i>satb2</i> is expressed robustly in the ventral pharyngeal arches and palatal precursors of wild-type embryos. (B) Expression of <i>satb2</i> in the palatal precursors and ventral arch 1 is retained in <i>sox32</i> mutants; however, <i>satb2</i> expression is ablated in the posterior arches.</p

Expression of <i>shh</i> and <i>satb2</i> correlate spatially during pharyngeal arch development.

<p>(A–D) Dorsal-lateral views of 30 hpf and 32 hpf wild-type embryos. (A) <i>shha</i> expression initiates in the pharyngeal endoderm at 30 hpf while (B) <i>satb2</i> is not expressed in the craniofacial region at this time. (C, arrows) By 32 hpf <i>shh</i> expression has intensified and can be seen to arc around the presumptive arches. (D, arrows) <i>satb2</i> expression is initiated in the ventral arch by 32 hpf and lies in close proximity to the presumptive pharyngeal endoderm.</p

Bmp signaling is required during early arch patterning for the appropriate expression of <i>satb2</i>.

<p>(A, C, E) Lateral and (B, D, F) ventral images of 36 hpf embryos labelled with <i>satb2</i> riboprobe. (A) The ventral region of the pharyngeal arches expresses <i>satb2</i> robustly. (B) In the ventral arches, <i>satb2</i> expression is strongest medially. (C, D) Inhibiting Bmp signaling <i>via</i> dorsomorphin from 20 to 36 hpf eliminates the majority of <i>satb2</i> expression including that in the palatal precursors (arrowhead in C). Only a small population of crest in ventromedial arch 2 expresses <i>satb2</i> following this treatment (arrows in C & D). (E, F) When Bmp signaling is blocked from 20 to 30 hpf and allowed to recover until 36 hpf, <i>satb2</i> expression is still greatly reduced. Palatal precursors fail to express <i>satb2</i> (arrowhead in E) and only ventromedial crest in arches 1 & 2 express <i>satb2</i> (arrows in E & F). Later inhibition of Bmp signaling does not alter <i>satb2</i> expression. (G–J) 42 hpf embryos labelled with riboprobe to <i>satb2</i> in lateral (G, I) and ventral (H, J) views. In both control embryos (G, H) and embryos treated with dorsomorphin from 30 to 42 hpf (I, J), <i>satb2</i> is strongly expressed by the palatal precursors and in the ventral pharyngeal arches. l, lateral; m, medial; p, palatal precursors.</p

Continuous Hh signaling is required for <i>satb2</i> expression.

<p>Ventral (A, B, I, J, M, N, Q, R), lateral (C, D, E, F, K, L, O, P, S, T, U, V) and dorsal-lateral (G, H) images of <i>satb2</i> expression by <i>in situ</i> hybridization are shown in control and cyclopmaine-treated (CYA) embryos. (A, C) In controls at 60 hpf, <i>satb2</i> is expressed in palatal precursors and in the ventral region of each arch. (B, D) Embryos treated with cyclopamine from 30–60 hpf show a dramatic reduction of <i>satb2</i> expression in the palatal precursors, pharyngeal arch 1 and 2 as well as complete loss of expression in the posterior arches. (E–H) Compared to controls, embryos treated with cyclopamine from 30 to 36 hpf also show reduction of <i>satb2</i> expression in the palatal precursors and arches 1 and 2, with a more complete loss of expression in the more posterior arches. (I–L) If, however, embryos are removed from cyclopamine at 36 hpf and allowed to develop for 10 hours, there is a partial recovery of <i>satb2</i> expression. (M–T) Cyclopamine treatment either from 38 to 42 hpf (M–P) or 44 to 50 hpf (Q–T) results in mild reduction of <i>satb2</i> expression in the palatal precursors and pharyngeal arches. (U, V) While the maxillary domain is lost in embryos treated with cyclopamine from 6–30 hpf the expression in the ventral arches appears largely intact. CYA, cyclopamine; e, eye; b, brain; p, palatal precursors; pharyngeal arches are numbered in some panels for clarity.</p