Lifecycle, culture, and maintenance of the emerging cephalopod models Euprymna berryi and Euprymna morsei

Cephalopod research remains limited by the inability to culture species under laboratory conditions for multiple generations to provide continuous access to animals at all stages of the life cycle. Here, we describe a multi-generational laboratory culture system for two emerging cephalopod models: the hummingbird or Berry’s bobtail squid, Euprymna berryi Sasaki, 1929, and Morse’s bobtail squid, Euprymna morsei Verrill, 1881, which are primarily found off mainland Japan. E. berryi wild adults were spawned and raised to the third filial generation, and E. morsei wild adults were spawned and raised to the second filial generation in a closed system at 20°C. We report growth and survivorship data for a cohort of 30 individuals across the first generation raised in captivity. E. berryi and E. morsei grew exponentially during the first 90 and 60 days post-hatching, respectively. Survivorship at the first spawning event for E. berryi and E. morsei was 90% and 77%. E. berryi and E. morsei females spawned after days 112 and 71 days post-hatching, respectively. We describe the life history of each species and how to distinguish sexes. We discuss the challenges of cephalopod culture and how culturing these species address those problems

From Pioneer to Repressor: Bimodal foxd3 Activity Dynamically Remodels Neural Crest Regulatory Landscape In Vivo

The neural crest (NC) is a transient embryonic stem cell-like population characterized by its multipotency and broad developmental potential. Here, we perform NC-specific transcriptional and epigenomic profiling of foxd3-mutant cells in vivo to define the gene regulatory circuits controlling NC specification. Together with global binding analysis obtained by foxd3 biotin-ChIP and single cell profiles of foxd3-expressing premigratory NC, our analysis shows that, during early steps of NC formation, foxd3 acts globally as a pioneer factor to prime the onset of genes regulating NC specification and migration by re-arranging the chromatin landscape, opening cis-regulatory elements and reshuffling nucleosomes. Strikingly, foxd3 then gradually switches from an activator to its well-described role as a transcriptional repressor and potentially uses differential partners for each role. Taken together, these results demonstrate that foxd3 acts bimodally in the neural crest as a switch from "permissive" to "repressive" nucleosome and chromatin organization to maintain multipotency and define cell fates

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

The Light Responsive Transcriptome of the Zebrafish: Function and Regulation

Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or “entrained” by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression

Thyrotroph Embryonic Factor Regulates Light-Induced Transcription of Repair Genes in Zebrafish Embryonic Cells

Numerous responses are triggered by light in the cell. How the light signal is detected and transduced into a cellular response is still an enigma. Each zebrafish cell has the capacity to directly detect light, making this organism particularly suitable for the study of light dependent transcription. To gain insight into the light signalling mechanism we identified genes that are activated by light exposure at an early embryonic stage, when specialised light sensing organs have not yet formed. We screened over 14,900 genes using micro-array GeneChips, and identified 19 light-induced genes that function primarily in light signalling, stress response, and DNA repair. Here we reveal that PAR Response Elements are present in all promoters of the light-induced genes, and demonstrate a pivotal role for the PAR bZip transcription factor Thyrotroph embryonic factor (Tef) in regulating the majority of light-induced genes. We show that tefβ transcription is directly regulated by light while transcription of tefα is under circadian clock control at later stages of development. These data leads us to propose their involvement in light-induced UV tolerance in the zebrafish embryo

Active nuclear transcriptome analysis reveals inflammasome-dependent mechanism for early neutrophil response to Mycobacterium marinum

The mechanisms governing neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilise biotagging, a novel genome-wide profiling approach based on cell type-specific in vivo biotinylation in zebrafish to analyse the initial response of neutrophils to Mycobacterium marinum, a close genetic relative of M. tuberculosis used to model tuberculosis. Differential expression analysis following nuclear RNA-seq of neutrophil active transcriptomes reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, NLRC3 ortholog (wu: fb15h11) and il1β. Crispr/Cas9-mediated knockout of caspase b, which acts by proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterial infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial neutrophil response to mycobacterial infection. Further perturbation of the IEGs exposes egr3 as a key transcriptional regulator controlling il1β transcription

Evidence from oyster suggests an ancient role for Pdx in regulating insulin gene expression in animals

Hox and ParaHox genes encode transcription factors with similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. Here, we report evidence indicating that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is a likely direct target of Pdx in Pacific oyster adults. We show that one insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue. Transcriptomic comparison suggests that this tissue plays a similar role to the vertebrate pancreas. Using ATAC-seq and ChIP, we identify an upstream regulatory element of the cgILP gene which shows binding interaction with cgPdx protein in oyster hepatopancreas and demonstrate, using a cell culture assay, that the oyster Pdx can act as a transcriptional activator through this site, possibly in synergy with NeuroD. These data argue that a classic homeodomain-target gene interaction dates back to the origin of Bilateria

Generation of a double binary transgenic zebrafish model to study myeloid gene regulation in response to oncogene activation in melanocytes

A complex network of inflammatory genes is closely linked to somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. Here, we describe the development of a double binary zebrafish model that enables regulatory programming of the myeloid cells as they respond to oncogene-activated melanocytes to be explored, focussing on the initial phase when cells become the precursors of cancer. A hormone-inducible binary system allows for temporal control of expression of different Ras oncogenes (NRasQ61K, HRasG12V and KRasG12V) in melanocytes, leading to proliferation and changes in morphology of the melanocytes. This model was coupled to binary cell-specific biotagging models allowing in vivo biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed as they respond to the earliest precursors of melanoma in vivo, revealed an intricate landscape of regulatory factors that may promote progression to melanoma, including Serpinb1l4, Fgf1, Fgf6, Cathepsin H, Galectin 1 and Galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma

A genome-wide assessment of the ancestral neural crest gene regulatory network

The neural crest (NC) is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional and epigenomic profiles of NC cells in the sea lamprey, in order to gain insight into the ancestral state of the NC gene regulatory network (GRN). Transcriptome analyses identify clusters of co-regulated genes during NC specification and migration that show high conservation across vertebrates but also identify transcription factors (TFs) and cell-adhesion molecules not previously implicated in NC migration. ATAC-seq analysis uncovers an ensemble of cis-regulatory elements, including enhancers of Tfap2B, SoxE1 and Hox-alpha2 validated in the embryo. Cross-species deployment of lamprey elements identifies the deep conservation of lamprey SoxE1 enhancer activity, mediating homologous expression in jawed vertebrates. Our data provide insight into the core GRN elements conserved to the base of the vertebrates and expose others that are unique to lampreys