Evolutionary changes in the notochord genetic toolkit: a comparative analysis of notochord genes in the ascidian Ciona and the larvacean Oikopleura

<p>Abstract</p> <p>Background</p> <p>The notochord is a defining feature of the chordate clade, and invertebrate chordates, such as tunicates, are uniquely suited for studies of this structure. Here we used a well-characterized set of 50 notochord genes known to be targets of the notochord-specific Brachyury transcription factor in one tunicate, <it>Ciona intestinalis </it>(Class Ascidiacea), to begin determining whether the same genetic toolkit is employed to build the notochord in another tunicate, <it>Oikopleura dioica </it>(Class Larvacea). We identified <it>Oikopleura </it>orthologs of the <it>Ciona </it>notochord genes, as well as lineage-specific duplicates for which we determined the phylogenetic relationships with related genes from other chordates, and we analyzed their expression patterns in <it>Oikopleura </it>embryos.</p> <p>Results</p> <p>Of the 50 <it>Ciona </it>notochord genes that were used as a reference, only 26 had clearly identifiable orthologs in <it>Oikopleura</it>. Two of these conserved genes appeared to have undergone <it>Oikopleura</it>- and/or tunicate-specific duplications, and one was present in three copies in <it>Oikopleura</it>, thus bringing the number of genes to test to 30. We were able to clone and test 28 of these genes. Thirteen of the 28 <it>Oikopleura </it>orthologs of <it>Ciona </it>notochord genes showed clear expression in all or in part of the <it>Oikopleura </it>notochord, seven were diffusely expressed throughout the tail, six were expressed in tissues other than the notochord, while two probes did not provide a detectable signal at any of the stages analyzed. One of the notochord genes identified, <it>Oikopleura netrin</it>, was found to be unevenly expressed in notochord cells, in a pattern reminiscent of that previously observed for one of the <it>Oikopleura </it><it>Hox </it>genes.</p> <p>Conclusions</p> <p>A surprisingly high number of <it>Ciona </it>notochord genes do not have apparent counterparts in <it>Oikopleura</it>, and only a fraction of the evolutionarily conserved genes show clear notochord expression. This suggests that <it>Ciona </it>and <it>Oikopleura</it>, despite the morphological similarities of their notochords, have developed rather divergent sets of notochord genes after their split from a common tunicate ancestor. This study demonstrates that comparisons between divergent tunicates can lead to insights into the basic complement of genes sufficient for notochord development, and elucidate the constraints that control its composition.</p

Temporal Regulation of the Muscle Gene Cascade by Macho1 and Tbx6 Transcription Factors in Ciona Intestinalis

For over a century, muscle formation in the ascidian embryo has been representative of \u27mosaic\u27 development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Brachyury, Foxa2 and the cis-Regulatory Origins of the Notochord

A main challenge of modern biology is to understand how specific constellations of genes are activated to differentiate cells and give rise to distinct tissues. This study focuses on elucidating how gene expression is initiated in the notochord, an axial structure that provides support and patterning signals to embryos of humans and all other chordates. Although numerous notochord genes have been identified, the regulatory DNAs that orchestrate development and propel evolution of this structure by eliciting notochord gene expression remain mostly uncharted, and the information on their configuration and recurrence is still quite fragmentary. Here we used the simple chordate Ciona for a systematic analysis of notochord cis-regulatory modules (CRMs), and investigated their composition, architectural constraints, predictive ability and evolutionary conservation. We found that most Ciona notochord CRMs relied upon variable combinations of binding sites for the transcription factors Brachyury and/or Foxa2, which can act either synergistically or independently from one another. Notably, one of these CRMs contains a Brachyury binding site juxtaposed to an (AC) microsatellite, an unusual arrangement also found in Brachyury-bound regulatory regions in mouse. In contrast, different subsets of CRMs relied upon binding sites for transcription factors of widely diverse families. Surprisingly, we found that neither intra-genomic nor interspecific conservation of binding sites were reliably predictive hallmarks of notochord CRMs. We propose that rather than obeying a rigid sequence-based cis-regulatory code, most notochord CRMs are rather unique. Yet, this study uncovered essential elements recurrently used by divergent chordates as basic building blocks for notochord CRMs

Engaging New Audiences with Imaging and Microscopy

In this Spotlight, we hear first-hand accounts from five scientists and educators who use microscopy and imaging to engage, entertain, educate and inspire new audiences with science and the field of developmental biology in particular. The \u27voices\u27 that follow each convey each authors\u27 own personal take on why microscopy is such a powerful tool for capturing the minds, and the hearts, of scientists, students and the public alike. They discuss how microscopy and imaging can reveal new worlds, and improve our communication and understanding of developmental biology, as well as break down barriers and promote diversity for future generations of scientific researchers

A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (K(d) 0.02 μM) compared with ATP-G-actin (K(d) 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved β-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly

Alternative regulatory mechanisms of notochord CRMs requiring Ci-Bra and/or Ci-Fox binding sites.

<p><b>a,f,m:</b> (Left) Schematic representations of wild-type (WT) and site-directed mutant CRMs; TF binding sites are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.g001" target="_blank">Fig 1</a>, with the mutant sequences indicated at the bottom of each panel. Mutated binding sites are colored in white and covered by “X” signs. Maroon bars represent constructs able to elicit notochord expression, while configurations exhibiting weak or no notochord staining are depicted by yellow and gray bars, respectively. (Right) Quantification of the fraction of the total stained embryos showing notochord expression after electroporation of the constructs at the left of each bar. n: number of fully developed stained embryos. Error bars denote standard deviation from the mean. <b>b-e, g-l, n-s:</b> Microphotographs of embryos expressing the transgenes indicated at the bottom right of each panel. Arrowheads are color-coded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.g001" target="_blank">Fig 1</a>. Abbreviations: WT: wild-type, F: Fox binding site, B: Brachyury binding site, HD: homeodomain, AP1: activator protein 1, mut: mutated, noto: notochord. In <b>f</b>, “S” stands for C/G. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.s002" target="_blank">S2 Fig</a>.</p

The function of individual Ci-Bra binding sites can be modulated by either an (AC) microsatellite or a flanking sequence.

<p><b>a,c:</b> Schematic representations of wild-type (WT) and mutant CRMs, as described and colored in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.g002" target="_blank">Fig 2</a>; the (AC) microsatellite sequence is schematized as a segmented brown rectangle. <b>b:</b> Mutational series of the area boxed in orange in the 253-bp construct. Red and blue nucleotides correspond to the Ci-Bra and Ci-Fox sites, respectively, and orange nucleotides indicate the bases changed in each mutant plasmid. The (AC)₆ microsatellite sequence is boxed in green. The relative ability of each construct to direct notochord gene expression is shown by plus signs at the right of each sequence. <b>d-i:</b> Photos of embryos electroporated with the constructs depicted in <b>a</b>,<b>b</b>,<b>c</b>; arrowheads are color-coded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.g001" target="_blank">Fig 1</a>. <b>j:</b> Quantification of notochord-stained embryos harboring the constructs in <b>a</b>,<b>c</b>. Error bars indicate standard deviation from the mean. <b>k:</b> Identification of an extended CTAM sequence (colored) shared by a subset of individually-acting Ci-Bra binding sites. <b>l-w:</b> Microphotographs of embryos carrying wild-type CRMs (<b>l,p,t</b>) compared to embryos carrying various mutant versions of Ci-CRM109 (<b>m-o</b>) Ci-CRM99 (<b>q-s</b>) and Ci-CRM86 (<b>u-w</b>). Core Ci-Bra binding sites are capitalized. Mutations are depicted in red. Abbreviations: FSM: “frame-shift” mutation, LSM: linker scanning mutation. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.s003" target="_blank">S3 Fig</a>.</p

A comparative study of notochord CRMs in <i>Ciona</i>.

<p><b>a-n:</b> Microphotographs of transgenic <i>Ciona</i> embryos expressing the <i>LacZ</i> reporter in the notochord (red arrowheads) under the control of 14 CRMs. (Right) Schematic representations of the 14 minimal notochord CRMs. Putative transcription factor binding sites are mapped along the length of each enhancer (tan bar), as indicated in the key (bottom). Point mutations uncovered site(s) required for notochord expression (colored and opaque) as well as sites that did not evidently contribute (colored, but transparent). Putative binding sites deemed dispensable through truncations are colored and hatched. Untested putative sites are outlined in gray. Additional staining domains are indicated by arrowheads, colored as follows: blue: CNS, yellow: endoderm, orange: muscle, purple: mesenchyme, green: epidermis. Embryos are oriented with dorsal up and anterior to the left. Scale bar: 40 μm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.s001" target="_blank">S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.s002" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005730#pgen.1005730.s003" target="_blank">S3</a> Figs</p