Immune function of the serosa in hemimetabolous insect eggs

Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria. For Oncopeltus, RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays, and describe two novel AMP families called Serosins and Ovicins. For both insects, qPCRs show immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects.

Single-nucleus RNA-sequencing in pre-cellularization Drosophila melanogaster embryos

Our current understanding of the regulation of gene expression in the early Drosophila melanogaster embryo comes from observations of a few genes at a time, as with in situ hybridizations, or observation of gene expression levels without regards to patterning, as with RNA-sequencing. Single-nucleus RNA-sequencing however, has the potential to provide new insights into the regulation of gene expression for many genes at once while simultaneously retaining information regarding the position of each nucleus prior to dissociation based on patterned gene expression. In order to establish the practicality of single-nucleus RNA sequencing in the context of a real biological question, here we look at the difference in gene expression between control and an insulator protein, dCTCF, maternal null embryos during zygotic genome activation at nuclear cycle 14. We find that early embryonic nuclei can be grouped into distinct clusters according to gene expression. From both virtual and published in situ hybridizations, we also find that these clusters correspond to spatial regions of the embryo. Lastly, we present multiple examples of differential gene expression between control and maternal CTCF null nuclei in one or more clusters, but not in bulk when grouping expression across all nuclei. These results highlight the potential for single-nucleus RNA-sequencing to reveal new insights into the regulation of gene expression in the early Drosophila melanogaster embryo.

optix is involved in eyespot development via a possible positional information mechanism

optix, a gene essential and sufficient for eye development in Drosophila melanogaster, also plays important roles in the development of both the structure and pigmentation of butterfly wing scales. In particular, optix regulates wing scale lower lamina thickness and ommochrome pigment synthesis. Here we explore the role of optix in wing pattern development of Bicyclus anynana butterflies by examining its expression using immunostainings and testing its function via CRISPR-Cas9. We found Optix to be expressed in multiple domains, most prominently in the orange ring of the eyespots and in other scattered orange scales, and to regulate the pigmentation and the development of the upper lamina of the orange scales. We further explored the interaction of Optix with Spalt, a protein involved in the development of black scales in the eyespots, and expressed adjacent to the Optix domain. CRISPR knockouts of optix or spalt, followed by immunostainings, showed that Spalt represses optix expression in cells of the central black region of the eyespot. This regulatory interaction mimics that found in the anterior compartment of the wing disc where both genes respond to Decapentaplegic (Dpp) signaling and play a role in venation patterning. Using in situ hybridizations we show that dpp is expressed in the center of the eyespots and propose that this same circuit might have been recruited for eyespot development where Decapentaplegic acts as a central morphogen, activating optix and spalt at different concentration thresholds, and where spalt cross-regulates optix resulting in the formation of a sharp boundary between the two eyespot color rings.

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Life at the Dallol geothermal area, Ethiopia- a Planetary Field Analog on Earth

<p><strong>Introduction</strong></p><p>Dallol volcano area, proposed as a Mars analog, is considered one of the most extreme environment on Earth. It is located in the Danakil depression, a tectonically and volcanically active region in Ethiopia. The hydrothermal springs that compose the system discharge hyperacidic hot Fe-rich brines, which results in halite deposition when they contact with the atmosphere. As a result, Dallol geothermal area shows extremely high temperature (90-108°C in the springs), pH from -1.7 to 4, and supersaturated salt deposits [1-3].</p><p>Still, <em>life finds a way</em>. Despite these extreme features, microorganisms inhabiting Dallol geothermal region, as members of the class Nanohaloarchaea, have been described [3,4]. The analysis of the biodiversity that are surviving and thriving in such polyextreme system, as well as the study of their metabolic potential, could be fundamental to establish the habitability conditions to search for life on other planets.</p><p><strong>Methods</strong></p><p>We applied Shotgun metagenomics and Fluorescence in situ Hybridizations (FISH) methods to analyze the biodiversity and metabolic potential of Dallol geothermal area microbiome.</p><p><strong>Results</strong></p><p>Our results show the presence of both bacteria and archaea in the Dallol geothermal area, being Bacteria the most abundant domain in this environment. The detected microorganisms are mostly halophiles. However, there is a lower percentage of microorganisms described in extremely hot or acidic environments. FISH experiments corroborated that both bacteria and archaea are alive and active in the system.</p><p>Metagenomics analysis shows a predominance of metabolisms based on photosynthesis, heterotrophy and fermentation. In addition, metabolisms that use hydrogen and reduced sulfur compounds as an energy source stand out.</p><p>Additionally, genes related to the response to oxidative stress, heat shock, osmotic stress and detoxification have been detected, which could be involved in the adaptation of these microorganisms to the extreme conditions of the Dallol volcano area.</p><p><strong>References</strong></p><p>[1] Cavalazzi, B., et al. (2019). The Dallol geothermal area, Northern Afar (Ethiopia)—An exceptional planetary field analog on Earth. Astrobiology, 19(4), 553-578.</p><p>[2] Kotopoulou, E., et al. (2018). A polyextreme hydrothermal system controlled by iron: the case of Dallol at the Afar Triangle. ACS Earth and Space Chemistry, 3(1), 90-99.</p><p>[3] Carrizo, D., et al. (2019). Lipid Biomarker and Carbon Stable Isotope Survey on the Dallol Hydrothermal System in Ethiopia. Astrobiology, 19(12), 1474-1489.</p><p>[4] Gómez, F., et al. (2019). Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia. Scientific reports, 9(1), 1-9.</p>

5-Hydroxytryptamine (5-HT) Positively Regulates Pigmentation via Inducing Melanoblast Specification and Melanin Synthesis in Zebrafish Embryos

It has been reported that 5-hydroxytryptamine (5-HT) is related to melanogenesis in mice and melanoma cells. However, the underlying mechanisms of 5-HT in regulating pigmentation remains unknown. In this study, we aim to clarify the regulatory mechanism of 5-HT in the pigmentation of zebrafish embryos and B16F10 cells. Our results show that 5-HT induces the pigmentation of zebrafish embryos in a dosage-dependent manner at concentrations of 0.01–1 mM. Whole mount in situ hybridizations and qRT-PCR in zebrafish embryos indicate that the expression of neural crest cells marker gene sox10 is not changed in embryos treated with 5-HT compared to control group. The expression of mitfa, the marker gene of melanoblasts, is increased in the presence of 5-HT. Furthermore, 5-HT increased the expression of regeneration associated genes, namely kita, mitfa, and dct, after ablation of the melanogenic cells in zebrafish embryos. The experiments in B16F10 cells show that 5-HT promotes melanin synthesis by up-regulating the expression of key proteins MITF, TYR, TRP-1, and TRP-2. Especially, the small molecule inhibitor of PKA signaling, but not AKT and MAPK signaling, attenuates the up-regulation of MITF and TYR resulted from 5-HT induction in B16F10 cells. These results will help us to further understand the regulatory network of vertebrate pigmentation.

Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos

Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

BgeeDB, an R package for retrieval of curated expression datasets and for gene list expression localization enrichment tests

BgeeDB is a collection of functions to import into R re-annotated, quality-controlled and re-processed expression data available in the Bgee database. This includes data from thousands of wild-type healthy samples of multiple animal species, generated with different gene expression technologies (RNA-seq, Affymetrix microarrays, expressed sequence tags, and in situ hybridizations). BgeeDB facilitates downstream analyses, such as gene expression analyses with other Bioconductor packages. Moreover, BgeeDB includes a new gene set enrichment test for preferred localization of expression of genes in anatomical structures (“TopAnat”). Along with the classical Gene Ontology enrichment test, this test provides a complementary way to interpret gene lists. Availability: https://www.bioconductor.org/packages/BgeeDB/