posterior end mark 2 (pem-2),pem-4,pem-5, andpem-6: Maternal Genes with Localized mRNA in the Ascidian Embryo

AbstractThe posterior–vegetal cytoplasm of an ascidian egg contains maternal factors required for pattern formation and cell specification of the embryo. We report here the isolation and characterization of cDNA clones for novel maternal genes,posterior end mark 2(pem-2),pem-4,pem-5, andpem-6. We obtained these clones from a cDNA library ofCiona savignyifertilized egg mRNAs subtracted with gastrula mRNAs by examining the localization of the corresponding mRNAs of randomly selected clones by whole-mountin situhybridization. As in the case ofpem, all of these mRNAs were localized in the posterior–vegetal cytoplasm of the egg, and they later marked the posterior end of early embryos. The predicted amino acid sequence suggested that PEM-2 contains a signal for nuclear localization, an src homology 3 (SH3) domain, and a consensus sequence of the CDC24 family guanine nucleotide dissociation stimulators (GDSs). PEM-4 has a signal for nuclear localization and three C2H2-type zinc finger motifs, while PEM-5 and PEM-6 show no similarity to known proteins. These results provide further evidence that the ascidian egg contains maternal messages that are localized in the posterior–vegetal cytoplasm

A digital twin reproducing gene regulatory network dynamics of early Ciona embryos indicates robust buffers in the network

How gene regulatory networks (GRNs) encode gene expression dynamics and how GRNs evolve are not well understood, although these problems have been studied extensively. We created a digital twin that accurately reproduces expression dynamics of 13 genes that initiate expression in 32-cell ascidian embryos. We first showed that gene expression patterns can be manipulated according to predictions by this digital model. Next, to simulate GRN rewiring, we changed regulatory functions that represented their regulatory mechanisms in the digital twin, and found that in 55 of 100 cases, removal of a single regulator from a conjunctive clause of Boolean functions did not theoretically alter qualitative expression patterns of these genes. In other words, we found that more than half the regulators gave theoretically redundant temporal or spatial information to target genes. We experimentally substantiated that the expression pattern of Nodal was maintained without one of these factors, Zfpm, by changing the upstream regulatory sequence of Nodal. Such robust buffers of regulatory mechanisms may provide a basis of enabling developmental system drift, or rewiring of GRNs without changing expression patterns of downstream genes, during evolution

A bHLH transcription factor gene, Twist-like1, is essential for the formation of mesodermal tissues of Ciona juveniles

AbstractAscidian larval mesenchyme cells, comprising about 900 cells, are derived from the A7.6, B8.5 and B7.7 blastomere pairs in the 110-cell embryo. Previous studies showed that the properties of mesenchyme cells are not uniform among the three lines in embryos of Ciona savignyi and Ciona intestinalis. After metamorphosis, the larval mesenchyme cells form the mesodermal tissues or organs of the adult body. In the present study, the developmental fates of A7.6-, B8.5- and B7.7-line mesenchyme cells were traced using DiI to determine the origins of juvenile mesodermal tissues of C. savignyi. It was demonstrated that each of the A7.6-, B8.5- and B7.7-line mesenchyme cells is distributed in different positions of the larval trunk, and then give rise to the different mesodermal tissues of juveniles. Twist-like1 is a transcription factor gene essential for the specification of larval mesenchyme cells. Knockdown of this gene with specific morpholino antisense oligonucleotides affected not only the specification of larval mesenchyme cells, but also the formation of most of the mesodermal tissues of juveniles. The juvenile mesodermal tissues in the Twist-like1-knockdown specimen were never compensated by the surrounding tissues. The present results therefore indicate that Twist-like1 is required for the differentiation of most mesodermal precursors of adults

A Twist-like bHLH gene is a downstream factor of an endogenous FGF and determines mesenchymal fate in the ascidian embryos

Ascidian larvae develop mesenchyme cells in their trunk. A fibroblast growth factor (FGF9/16/20) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. We have identified two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 as downstream factors of this FGF. These two genes are phylogenetically closely related to each other, and were expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrated that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream factor of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. We also demonstrate that the upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called `trunk lateral cells'. FGF9/16/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH factor called NoTrlc are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, which triggers the differentiation of the mesenchyme in Ciona embryos

The evolutionary conservation of the core components necessary for the extrinsic apoptotic signaling pathway, in Medaka fish

<p>Abstract</p> <p>Background</p> <p>Death receptors on the cell surface and the interacting cytosolic molecules, adaptors and initiator caspases, are essential as core components of the extrinsic apoptotic signaling pathway. While the apoptotic machinery governing the extrinsic signaling pathway is well characterized in mammals, it is not fully understood in fish.</p> <p>Results</p> <p>We identified and characterized orthologs of mammalian Fas, FADD and caspase-8 that correspond to the death receptor, adaptor and initiator caspase, from the Medaka fish (<it>Oryzias latipes</it>). Medaka Fas, caspase-8 and FADD exhibited protein structures similar to that of their mammalian counterparts, containing a death domain (DD), a death effector domain (DED) or both. Functional analyses indicated that these molecules possess killing activity in mammalian cell lines upon overexpression or following activation by apoptotic stimuli, suggesting similar pro-apoptotic functions in the extrinsic pathway as those in mammals. Genomic sequence analysis revealed that the Medaka <it>fas </it>(<it>tnfrsf6</it>), <it>fadd </it>and <it>caspase-8 </it>(<it>casp8</it>) genes are organized in a similar genomic structure as the mammalian genes. Database search and phylogenetic analysis revealed that the <it>fas </it>gene, but not the <it>fadd </it>and <it>casp8 </it>genes, appear to be present only in vertebrates.</p> <p>Conclusion</p> <p>Our results indicate that the core components necessary for the extrinsic apoptotic pathway are evolutionarily conserved in function and structure across vertebrate species. Based on these results, we presume the mechanism of apoptosis induction via death receptors was evolutionarily established during the appearance of vertebrates.</p

The gene regulatory system for specifying germ layers in early embryos of the simple chordate

動物胚の遺伝子発現を数式で表現 --動物の胚葉形成システムをまるごと理解--.京都大学プレスリリース. 2021-06-10.In animal embryos, gene regulatory networks control the dynamics of gene expression in cells and coordinate such dynamics among cells. In ascidian embryos, gene expression dynamics have been dissected at the single-cell resolution. Here, we revealed mathematical functions that represent the regulatory logics of all regulatory genes expressed at the 32-cell stage when the germ layers are largely specified. These functions collectively explain the entire mechanism by which gene expression dynamics are controlled coordinately in early embryos. We found that regulatory functions for genes expressed in each of the specific lineages contain a common core regulatory mechanism. Last, we showed that the expression of the regulatory genes became reproducible by calculation and controllable by experimental manipulations. Thus, these regulatory functions represent an architectural design for the germ layer specification of this chordate and provide a platform for simulations and experiments to understand the operating principles of gene regulatory networks

Genomic overview of mRNA 5′-leader trans-splicing in the ascidian Ciona intestinalis

Although spliced leader (SL) trans-splicing in the chordates was discovered in the tunicate Ciona intestinalis there has been no genomic overview analysis of the extent of trans-splicing or the make-up of the trans-spliced and non-trans-spliced gene populations of this model organism. Here we report such an analysis for Ciona based on the oligo-capping full-length cDNA approach. We randomly sampled 2078 5′-full-length ESTs representing 668 genes, or 4.2% of the entire genome. Our results indicate that Ciona contains a single major SL, which is efficiently trans-spliced to mRNAs transcribed from a specific set of genes representing ∼50% of the total number of expressed genes, and that individual trans-spliced mRNA species are, on average, 2–3-fold less abundant than non-trans-spliced mRNA species. Our results also identify a relationship between trans-splicing status and gene functional classification; ribosomal protein genes fall predominantly into the non-trans-spliced category. In addition, our data provide the first evidence for the occurrence of polycistronic transcription in Ciona. An interesting feature of the Ciona polycistronic transcription units is that the great majority entirely lack intercistronic sequences

Gata is ubiquitously required for the earliest zygotic gene transcription in the ascidian embryo

In ascidian embryos, the earliest transcription from the zygotic genome begins between the 8-cell and 16-cell stages. Gata.a, a maternally expressed Gata transcription factor, activates target genes specifically in the animal hemisphere, whereas the complex of β-catenin and Tcf7 antagonizes the activity of Gata.a and activates target genes specifically in the vegetal hemisphere. Here, we show that genes zygotically expressed at the 16-cell stage have significantly more Gata motifs in their upstream regions. These genes included not only genes with animal hemisphere-specific expression but also genes with vegetal hemisphere-specific expression. On the basis of this finding, we performed knockdown experiments for Gata.a and reporter assays, and found that Gata.a is required for the expression of not only genes with animal hemisphere-specific expression, but also genes with vegetal hemisphere-specific expression. Our data indicated that weak Gata.a activity that cannot induce animal hemisphere-specific expression can allow β-catenin/Tcf7 targets to be expressed in the vegetal cells. Because genes zygotically expressed at the 32-cell stage also had significantly more Gata motifs in their upstream regions, Gata.a function may not be limited to the genes expressed specifically in the animal or vegetal hemispheres at the 16-cell stage, and Gata.a may play an important role in the earliest transcription of the zygotic genome

Using linkage logic theory to control dynamics of a gene regulatory network of a chordate embryo.

Linkage logic theory provides a mathematical criterion to control network dynamics by manipulating activities of a subset of network nodes, which are collectively called a feedback vertex set (FVS). Because many biological functions emerge from dynamics of biological networks, this theory provides a promising tool for controlling biological functions. By manipulating the activity of FVS molecules identified in a gene regulatory network (GRN) for fate specification of seven tissues in ascidian embryos, we previously succeeded in reproducing six of the seven cell types. Simultaneously, we discovered that the experimentally reconstituted GRN lacked information sufficient to reproduce muscle cells. Here, we utilized linkage logic theory as a tool to find missing edges in the GRN. Then, we identified a FVS from an updated version of the GRN and confirmed that manipulating the activity of this FVS was sufficient to induce all seven cell types, even in a multi-cellular environment. Thus, linkage logic theory provides tools to find missing edges in experimentally reconstituted networks, to determine whether reconstituted networks contain sufficient information to fulfil expected functions, and to reprogram cell fate