Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling.

Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors (TFs), such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C. robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2.b and an inferred ATTA-binding transcriptional cofactor. Here, we show that the homeobox TF Lhx3/4 serves as the lineage-determining TF that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings, we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term "cofactor-dependent induction." Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.

The Ciona myogenic regulatory factor functions as a typical MRF but possesses a novel N-terminus that is essential for activity.

Electroporation-based assays were used to test whether the myogenic regulatory factor (MRF) of Ciona intestinalis (CiMRF) interferes with endogenous developmental programs, and to evaluate the importance of its unusual N-terminus for muscle development. We found that CiMRF suppresses both notochord and endoderm development when it is expressed in these tissues by a mechanism that may involve activation of muscle-specific microRNAs. Because these results add to a large body of evidence demonstrating the exceptionally high degree of functional conservation among MRFs, we were surprised to discover that non-ascidian MRFs were not myogenic in Ciona unless they formed part of a chimeric protein containing the CiMRF N-terminus. Equally surprising, we found that despite their widely differing primary sequences, the N-termini of MRFs of other ascidian species could form chimeric MRFs that were also myogenic in Ciona. This domain did not rescue the activity of a Brachyury protein whose transcriptional activation domain had been deleted, and so does not appear to constitute such a domain. Our results indicate that ascidians have previously unrecognized and potentially novel requirements for MRF-directed myogenesis. Moreover, they provide the first example of a domain that is essential to the core function of an important family of gene regulatory proteins, one that, to date, has been found in only a single branch of the family.

Efficient genome editing using CRISPR-Cas-mediated homology directed repair in the ascidian Ciona robusta.

Eliminating or silencing a gene's level of activity is one of the classic approaches developmental biologists employ to determine a gene's function. A recently developed method of gene perturbation called CRISPR-Cas, which was derived from a prokaryotic adaptive immune system, has been adapted for use in eukaryotic cells. This technology has been established in several model organisms as a powerful and efficient tool for knocking out or knocking down the function of a gene of interest. It has been recently shown that CRISPR-Cas functions with fidelity and efficiency in Ciona robusta. Here, we show that in C. robusta CRISPR-Cas mediated genomic knock-ins can be efficiently generated. Electroporating a tissue-specific transgene driving Cas9 and a U6-driven gRNA transgene together with a fluorescent protein-containing homology directed repair (FP-HDR) template results in gene-specific patterns of fluorescence consistent with a targeted genomic insertion. Using the Tyrosinase locus to optimize reagents, we first characterize a new Pol III promoter for expressing gRNAs from the Ciona savignyi H1 gene, and then adapt technology that flanks gRNAs by ribozymes allowing cell-specific expression from Pol II promoters. Next, we examine homology arm-length efficiencies of FP-HDR templates. Reagents were then developed for targeting Brachyury and Pou4 that resulted in expected patterns of fluorescence, and sequenced PCR amplicons derived from single embryos validated predicted genomic insertions. Finally, using two differentially colored FP-HDR templates, we show that biallelic FP-HDR template insertion can be detected in live embryos of the F0 generation.

Acquisition of polymorphism in the chordate doliolids.

In polymorphic organisms a single genome is deployed to program numerous, morphologically distinct body plans within a colony. This complex life history trait has evolved independently within a limited subset of animal taxa. Reconstructing the underlying genetic, cellular and developmental changes that drove the emergence of polymorphic colonies represents a promising avenue for exploring diversifying selection and resulting impacts on developmental gene regulatory networks. Doliolids are the only polymorphic chordate, deploying a single genome to program distinct morphs specialized for locomotion, feeding, asexual or sexual reproduction. In this review, we provide a detailed summary of doliolid anatomy, development, taxonomy, ecology, life history and the cellular basis for doliolid polymorphism. In order to frame the potential evolutionary and developmental insights that could be gained by studying doliolids we provide a broader overview of polymorphism. We then discuss how comparative studies of polymorphic cnidarians have begun to illuminate the genetic basis of this unusual and complex life history strategy. We then provide a summary of life history divergence in the chordates, particularly among doliolids and their polymorphic cousins, the salps and pyrosomes.

Structure and vibrational dynamics of model compounds of the [FeFe]-hydrogenase enzyme system via ultrafast two-dimensional infrared spectroscopy.

Ultrafast two-dimensional infrared (2D) spectroscopy has been applied to study the structure and vibrational dynamics of (mu-S(CH2)3S)Fe2(CO)6, a model compound of the active site of the [FeFe]-hydrogenase enzyme system. Comparison of 2D-IR spectra of (mu-S(CH2)3S)Fe2(CO)6 with density functional theory calculations has determined that the solution-phase structure of this molecule is similar to that observed in the crystalline phase and in good agreement with gas-phase simulations. In addition, vibrational coupling and rapid (<5 ps) solvent-mediated equilibration of energy between vibrationally excited states of the carbonyl ligands of the di-iron-based active site model are observed prior to slower (approximately 100 ps) relaxation to the ground state. These dynamics are shown to be solvent-dependent and form a basis for the future determination of the vibrational interactions between active site and protein.

Electrocatalytic proton reduction by dithiolate-bridged diiron carbonyl complexes: a connection to the H-cluster?

Spectroscopic and electrochemical investigation of electrocatalytic proton reduction by Fe(2)(mu-pdt)(CO)(6), 1, have been interpreted in terms of a reaction scheme involving sequential electron-proton reactions to give a two-electron, two-proton product that undergoes rate-limiting dihydrogen elimination. Further reduction, at slightly higher negative potentials, gives a more reactive product and this process dominates reactions conducted at higher acid concentrations. Inhibition of the electrocatalytic reaction by CO is due to the more efficient loss of catalyst and this is best modelled by a reaction that is second order in terms of 1(-). During electrocatalytic proton reduction a new species is observed, which features a bridging CO group and the wavenumbers of the nu(CO) modes of the terminally bound carbonyl groups are similar to those of the carbonyl groups bound to the oxidized form of the H-cluster.

Polyferredoxin-based electrode materials.

Electrochemical oxidation of the hydrosulfide cluster [Fe4S4(SH)4]2- on gold, platinum or vitreous carbon in a methyl cyanide electrolyte leads to the growth of a conducting film. Spectroscopic and other evidence suggests that the film has cubane centres, predominately in the [4Fe4S]3+ oxidation state, which are linked by disulfide ligands to give an anionic array of [Fe4S4(S approximately)4]n- units. X-ray data suggests some long-range order in the electrode material. The polyferredoxin binds redox active cations consistent with an anionic array.