Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Xbp1 and Brachyury establish an evolutionarily conserved subcircuit of the notochord gene regulatory network.

Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.

Transcription Factors of the bHLH Family Delineate Vertebrate Landmarks in the Nervous System of a Simple Chordate.

Tunicates are marine invertebrates whose tadpole-like larvae feature a highly simplified version of the chordate body plan. Similar to their distant vertebrate relatives, tunicate larvae develop a regionalized central nervous system and form distinct neural structures, which include a rostral sensory vesicle, a motor ganglion, and a caudal nerve cord. The sensory vesicle contains a photoreceptive complex and a statocyst, and based on the comparable expression patterns of evolutionarily conserved marker genes, it is believed to include proto-hypothalamic and proto-retinal territories. The evolutionarily conserved molecular fingerprints of these landmarks of the vertebrate brain consist of genes encoding for different transcription factors, and of the gene batteries that they control, and include several members of the bHLH family. Here we review the complement of bHLH genes present in the streamlined genome of the tunicate Ciona robusta and their current classification, and summarize recent studies on proneural bHLH transcription factors and their expression territories. We discuss the possible roles of bHLH genes in establishing the molecular compartmentalization of the enticing nervous system of this unassuming chordate.

Enhancer of Polycomb and the Tip60 complex repress hematological tumor initiation by negatively regulating JAK/STAT pathway activity.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders that cause excessive production of myeloid cells. Most MPN patients have a point mutation in JAK2 (JAK2V617F ), which encodes a dominant-active kinase that constitutively triggers JAK/STAT signaling. In Drosophila, this pathway is simplified, with a single JAK, Hopscotch (Hop), and a single STAT transcription factor, Stat92E. The hopTumorous-lethal [hopTum ] allele encodes a dominant-active kinase that induces sustained Stat92E activation. Like MPN patients, hopTum mutants have significantly more myeloid cells, which form invasive tumors. Through an unbiased genetic screen, we found that heterozygosity for Enhancer of Polycomb [E(Pc)], a component of the Tip60 lysine acetyltransferase complex (also known as KAT5 in humans), significantly increased tumor burden in hopTum animals. Hematopoietic depletion of E(Pc) or other Tip60 components in an otherwise wild-type background also induced blood cell tumors. The E(Pc) tumor phenotype was dependent on JAK/STAT activity, as concomitant depletion of hop or Stat92E inhibited tumor formation. Stat92E target genes were significantly upregulated in E(Pc)-mutant myeloid cells, indicating that loss of E(Pc) activates JAK/STAT signaling. Neither the hop nor Stat92E gene was upregulated upon hematopoietic E(Pc) depletion, suggesting that the regulation of the JAK/STAT pathway by E(Pc) is dependent on substrates other than histones. Indeed, E(Pc) depletion significantly increased expression of Hop protein in myeloid cells. This study indicates that E(Pc) works as a tumor suppressor by attenuating Hop protein expression and ultimately JAK/STAT signaling. Since loss-of-function mutations in the human homologs of E(Pc) and Tip60 are frequently observed in cancer, our work could lead to new treatments for MPN patients.This article has an associated First Person interview with the first author of the paper.

Control of thiol-maleimide reaction kinetics in PEG hydrogel networks.

Michael-type addition reactions are widely used to polymerize biocompatible hydrogels. The thiol-maleimide modality achieves the highest macromer coupling efficiency of the reported Michael-type pairs, but the resulting hydrogel networks are heterogeneous because polymerization is faster than the individual components can be manually mixed. The reactivity of the thiol dictates the overall reaction speed, which can be slowed in organic solvents and acidic buffers. Since these modifications also reduce the biocompatibility of resulting hydrogels, we investigated a series of biocompatible buffers and crosslinkers to decelerate gelation while maintaining high cell viability. We found that lowering the polymer weight percentage (wt%), buffer concentration, and pH slowed gelation kinetics, but crosslinking with an electronegative peptide was optimal for both kinetics and cell viability. Including a high glucose medium supplement in the polymer solvent buffer improved the viability of the cells being encapsulated without impacting gelation time. Slowing the speed of polymerization resulted in more uniform hydrogels, both in terms of visual inspection and the diffusion of small molecules through the network. However, reactions that were too slow resulted in non-uniform particle dispersion due to settling, thus there is a trade-off in hydrogel network uniformity versus cell distribution in the hydrogels when using these networks in cell applications. STATEMENT OF SIGNIFICANCE: The polymer network of thiol-maleimide hydrogels assembles faster than individual components can be uniformly mixed due to their fast gelation kinetics. The lack of homogeneity can result in variable cell-based assay results, resulting in batch-to-batch variability and limiting their use in predictive screening assays. Although these hydrogels are incredibly useful in tissue engineering, this network heterogeneity is a known problem in the field. We screened a variety of possible techniques to slow down the reaction speed and improve the homogeneity of these hydrogels, without sacrificing the viability and distribution of encapsulated cells. As others have reported, an electronegative crosslinker was the most effective technique to slow the reaction, but the chemical modification required is technically challenging. Of interest to a broad community, we screened buffer type, strength, and crosslinker electronegativity to find an optimal reaction speed that allows for high cell viability and small molecule diffusion, without allowing cells to settle during gelation, allowing application of these materials to the drug screening industry and tissue engineering community.