Mapping and Functional Role of Phosphorylation Sites in the Thyroid Transcription Factor-1 (TTF-1)

The phosphorylation of thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor that is required for thyroid-specific expression of the thyroglobulin and thyroperoxidase gene promoters, has been studied. Phosphorylation occurs on a maximum of seven serine residues that are distributed in three tryptic peptides. Mutant derivatives of TTF-1, with alanine residues replacing the serines in the phosphorylation sites, have been constructed and used to assess the functional relevance of TTF-1 phosphorylation. The DNA binding activity of TTF-1 appears to be phosphorylation-independent, as indicated also by the performance of TTF-1 purified from an overexpressing Escherichia coli strain. Transcriptional activation by TTF-1 could require phosphorylation only in specific cell types since in a co-transfection assay in heterologous cells both wild-type and mutant proteins show a similar transcriptional activity

Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time polymerase chain reaction (RT-qPCR) is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc <it>Octopus vulgaris</it>, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization.</p> <p>Results</p> <p>We chose <it>16S</it>, and <it>18S </it>rRNA, <it>actB</it>, <it>EEF1A</it>, <it>tubA </it>and <it>ubi </it>as candidate reference genes (housekeeping genes, HKG). The expression of <it>16S </it>and <it>18S </it>was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts) and mantle (here considered as control tissue) by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed <it>tubA </it>and <it>ubi </it>as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes <it>FoxP</it>, <it>creb, dat </it>and <it>TH </it>in <it>O. vulgaris</it>.</p> <p>Conclusion</p> <p>We analyzed the expression profiles of some genes here identified for <it>O. vulgaris </it>by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of <it>ubi </it>and <it>tubA </it>to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the importance of choosing a proper normalization strategy when analyzing gene expression by qPCR taking into appropriate account the experimental setting and variability of the sample of animals (and tissues), thus providing a set of HGK which expression appears to be unaffected by the experimental factor(s).</p

Integrating single cell transcriptomics and volume electron microscopy confirms the presence of pancreatic acinar-like cells in sea urchins

The identity and function of a given cell type relies on the differential expression of gene batteries that promote diverse phenotypes and functional specificities. Therefore, the identification of the molecular and morphological fingerprints of cell types across taxa is essential for untangling their evolution. Here we use a multidisciplinary approach to identify the molecular and morphological features of an exocrine, pancreas-like cell type harbored within the sea urchin larval gut. Using single cell transcriptomics, we identify various cell populations with a pancreatic-like molecular fingerprint that are enriched within the S. purpuratus larva digestive tract. Among these, in the region where they reside, the midgut/stomach domain, we find that populations of exocrine pancreas-like cells have a unique regulatory wiring distinct from the rest the of the cell types of the same region. Furthermore, Serial Block-face scanning Electron Microscopy (SBEM) of the exocrine cells shows that this reported molecular diversity is associated to distinct morphological features that reflect the physiological and functional properties of this cell type. Therefore, we propose that these sea urchin exocrine cells are homologous to the well-known mammalian pancreatic acinar cells and thus we trace the origin of this particular cell type to the time of deuterostome diversification. Overall, our approach allows a thorough characterization of a complex cell type and shows how both the transcriptomic and morphological information contribute to disentangling the evolution of cell types and organs such as the pancreatic cells and pancreas. Keywords: SBEM; acinar cells; evolution of cell types; morphology; pancreas; scRNAseq; sea urchin

Whole animal freeze-fracture scanning electron microscopy: an easy-to-use method to investigate cell type morphology of marine embryos and larvae

Morphological and molecular characterization of cell types, organs and individual organisms is essential for understanding the origins of morphogenesis. The increased implementation of high throughput methods as a means to address cell type evolution, during the last decade, created the need for an efficient way to assess cell type morphology. Here in order to create a new tool to study cell type morphology, we optimized a fast and easy-to-use whole animal freeze-fracture scanning electron microscopy (WAFFSEM) method. This method was applied on marine experimental systems (echinoderms, mollusks, tunicates, and cephalochordates), that have been widely used to assess environmental, developmental, and evolutionary questions. Our protocol does not require any specialized equipment and the processed specimens are compatible with scanning electron microscopy. This protocol was able to successfully expose the internal cell types of all specimens in which it was tested and to reveal their cellular and subcellular characteristics. We strongly believe that the combination of our protocol with other methods (e.g., light microscopy and single cell transcriptomics) will be beneficial to further improve the way to classify and describe cell types

Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins

DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module

Developmental toxicity of plastic leachates on the sea urchin Paracentrotus lividus

Microplastic pollution has become ubiquitous, affecting a wide variety of biota. Although microplastics are known to alter the development of a range of marine invertebrates, no studies provide a detailed morphological characterisation of the developmental defects. Likewise, the developmental toxicity of chemicals leached from plastic particles is understudied. The consequences of these developmental effects are likely underestimated, and the effects on ecosystems are unknown. Using the sea urchin Paracentrotus lividus as a model, we studied the effects of leachates of three forms of plastic pellet: new industrial pre-production plastic nurdles, beached pre-production nurdles, and floating filters, known as biobeads, also retrieved from the environment. Our chemical analyses show that leachates from beached pellets (biobead and nurdle pellets) and highly plasticised industrial pellets (PVC) contain polycyclic aromatic hydrocarbons and polychlorinated biphenyls, which are known to be detrimental to development and other life stages of animals. We also demonstrate that these microplastic leachates elicit severe, consistent and treatment-specific developmental abnormalities in P. lividus at embryonic and larval stages. Those embryos exposed to virgin polyethylene leachates with no additives nor environmental contaminants developed normally, suggesting that the abnormalities observed are the result of exposure to either environmentally adsorbed contaminants or pre-existing industrial additives within the polymer matrix. In the light of the chemical contents of the leachates and other characteristics of the plastic particles used, we discuss the phenotypes observed during our study, which include abnormal gastrulation, impaired skeletogenesis, abnormal neurogenesis, redistribution of pigmented cells and embryo radialisation

Evolution of the ribbon-like organization of the Golgi apparatus in animal cells

The ‘‘ribbon,’’ a structural arrangement in which Golgi stacks connect to each other, is considered to be restricted to vertebrate cells. Although ribbon disruption is linked to various human pathologies, its functional role in cellular processes remains unclear. In this study, we investigate the evolutionary origin of the Golgi ribbon. We observe a ribbon-like architecture in the cells of several metazoan taxa suggesting its early emergence in animal evolution predating the appearance of vertebrates. Supported by AlphaFold2 modeling, we propose that the evolution of Golgi reassembly and stacking protein (GRASP) binding by golgin tethers may have driven the joining of Golgi stacks resulting in the ribbon-like configuration. Additionally, we find that Golgi ribbon assembly is a shared developmental feature of deuterostomes, implying a role in embryogenesis. Overall, our study points to the functional significance of the Golgi ribbon beyond vertebrates and underscores the need for further investigations to unravel its elusive biological roles

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement