BackgroundOrganoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined.ResultsHere we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask.ConclusionOur protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.

BackgroundMyopia (short-sightedness) affects approximately 1.4 billion people worldwide, and prevalence is increasing. Animal models induced by defocusing lenses show striking similarity with human myopia in terms of morphology and the implicated genetic pathways. Less is known about proteome changes in animals. Thus, the present study aimed to improve understanding of protein pathway responses to lens defocus, with an emphasis on relating expression changes to no lens control development and identifying bidirectional and/or distinct pathways across myopia and hyperopia (long-sightedness) models.ResultsQuantitative label-free proteomics and gene set enrichment analysis (GSEA) were used to examine protein pathway expression in the retina/RPE of chicks following 6 h and 48 h of myopia induction with − 10 dioptre (D) lenses, hyperopia induction with +10D lenses, or normal no lens rearing. Seventy-one pathways linked to cell development and neuronal maturation were differentially enriched between 6 and 48 h in no lens chicks. The majority of these normal developmental changes were disrupted by lens-wear (47 of 71 pathways), however, only 11 pathways displayed distinct expression profiles across the lens conditions. Most notably, negative lens-wear induced up-regulation of proteins involved in ATP-driven ion transport, calcium homeostasis, and GABA signalling between 6 and 48 h, while the same proteins were down-regulated over time in normally developing chicks. Glutamate and bicarbonate/chloride transporters were also down-regulated over time in normally developing chicks, and positive lens-wear inhibited this down-regulation.ConclusionsThe chick retina/RPE proteome undergoes extensive pathway expression shifts during normal development. Most of these pathways are further disrupted by lens-wear. The identified expression patterns suggest close interactions between neurotransmission (as exemplified by increased GABA receptor and synaptic protein expression), cellular ion homeostasis, and associated energy resources during myopia induction. We have also provided novel evidence for changes to SLC-mediated transmembrane transport during hyperopia induction, with potential implications for signalling at the photoreceptor-bipolar synapse. These findings reflect a key role for perturbed neurotransmission and ionic homeostasis in optically-induced refractive errors, and are predicted by our Retinal Ion Driven Efflux (RIDE) model.

BackgroundHoneybee development consists of four stages: embryo, larva, pupa and adult. Embryogenesis, a key process of cell division and differentiation, takes 3 days in honeybees. However, the embryonic transcriptome and the dynamic regulation of embryonic transcription are still largely uncharacterized in honeybees, especially in the Asian honeybee (Apis cerana). Here, we employed high-quality RNA-seq to explore the transcriptome of Asian honeybee embryos at three ages, approximately 24, 48 and 72 h (referred to as Day1, Day2 and Day3, respectively).ResultsNine embryo samples, three from each age, were collected for RNA-seq. According to the staging scheme of honeybee embryos and the morphological features we observed, our Day1, Day2 and Day3 embryos likely corresponded to the late stage four, stage eight and stage ten development stages, respectively. Hierarchical clustering and principal component analysis showed that same-age samples were grouped together, and the Day2 samples had a closer relationship with the Day3 samples than the Day1 samples. Finally, a total of 18,284 genes harboring 55,646 transcripts were detected in the A. cerana embryos, of which 44.5% consisted of the core transcriptome shared by all three ages of embryos. A total of 4088 upregulated and 3046 downregulated genes were identified among the three embryo ages, of which 2010, 3177 and 1528 genes were upregulated and 2088, 2294 and 303 genes were downregulated from Day1 to Day2, from Day1 to Day3 and from Day2 to Day3, respectively. The downregulated genes were mostly involved in cellular, biosynthetic and metabolic processes, gene expression and protein localization, and macromolecule modification; the upregulated genes mainly participated in cell development and differentiation, tissue, organ and system development, and morphogenesis. Interestingly, several biological processes related to the response to and detection of light stimuli were enriched in the first-day A. cerana embryogenesis but not in the Apis mellifera embryogenesis, which was valuable for further investigations.ConclusionsOur transcriptomic data substantially expand the number of known transcribed elements in the A. cerana genome and provide a high-quality view of the transcriptome dynamics of A. cerana embryonic development.

BackgroundOver one third of all animal phyla utilize a mode of early embryogenesis called ‘spiral cleavage’ to divide the fertilized egg into embryonic cells with different cell fates. This mode is characterized by a series of invariant, stereotypic, asymmetric cell divisions (ACDs) that generates cells of different size and defined position within the early embryo. Astonishingly, very little is known about the underlying molecular machinery to orchestrate these ACDs in spiral-cleaving embryos. Here we identify, for the first time, cohorts of factors that may contribute to early embryonic ACDs in a spiralian embryo.ResultsTo do so we analyzed stage-specific transcriptome data in eggs and early embryos of the spiralian annelid Platynereis dumerilii for the expression of over 50 candidate genes that are involved in (1) establishing cortical domains such as the partitioning defective (par) genes, (2) directing spindle orientation, (3) conveying polarity cues including crumbs and scribble, and (4) maintaining cell-cell adhesion between embryonic cells. In general, each of these cohorts of genes are co-expressed exhibiting high levels of transcripts in the oocyte and fertilized single-celled embryo, with progressively lower levels at later stages. Interestingly, a small number of key factors within each ACD module show different expression profiles with increased early zygotic expression suggesting distinct regulatory functions. In addition, our analysis discovered several highly co-expressed genes that have been associated with specialized neural cell-cell recognition functions in the nervous system. The high maternal contribution of these ‘neural’ adhesion complexes indicates novel general adhesion functions during early embryogenesis.ConclusionsSpiralian embryos are champions of ACD generating embryonic cells of different size with astonishing accuracy. Our results suggest that the molecular machinery for ACD is already stored as maternal transcripts in the oocyte. Thus, the spiralian egg can be viewed as a totipotent yet highly specialized cell that evolved to execute fast and precise ACDs during spiral cleaving stages. Our survey identifies cohorts of factors in P. dumerilii that are candidates for these molecular mechanisms and their regulation, and sets the stage for a functional dissection of ACD in a spiral-cleaving embryo.

BackgroundThe Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein.ResultsTo address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform.ConclusionsOur results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production.

BackgroundThe quality and yield of duck feathers are very important economic traits that might be controlled by miRNA regulation. The aim of the present study was to investigate the mechanism underlying the crosstalk between individual miRNAs and the activity of signaling pathways that control the growth of duck feathers during different periods. We therefore conducted a comprehensive investigation using Solexa sequencing technology on the Pekin duck microRNAome over six stages of feather development at days 11, 15, and 20 of embryonic development (during the hatching period), and at 1 day and 4 and 10 weeks posthatch.ResultsThere were a total of 354 known miRNAs and 129 novel candidate miRNAs found based on comparisons with known miRNAs in the Gallus gallus miRBase. The series of miRNAs related to feather follicle formation as summarized in the present study showed two expression patterns, with primary follicle developed during embryonic stage and secondary follicle developed mainly at early post hatch stage. Analysis of miRNA expression profiles identified 18 highly expressed miRNAs, which might be directly responsible for regulation of feather development. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that in addition to Wnt and transforming growth factor (TGFβ) signaling pathways, which were widely reported in response to follicle formation, another group of signaling pathways that regulate lipid synthesis and metabolism, such as the phosphatidylinositol signaling system and glycerolipid metabolism and signaling, are also responsible for follicle formation.ConclusionThe highly expressed miRNAs provide a valuable reference for further investigation into the functional miRNAs important for feather development. Lipid synthesis and metabolism related signaling pathways might be responsible for lipid formation on the surface of feather, and should be paid much more attention for their relation to feather quality.

BackgroundThe tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3Tg/Tg homozygotes are not viable and TgTP6.3Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3.ResultsAlthough TgTP6.4Tg/Tg homozygotes died before weaning, TgTP6.4Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex.ConclusionsMosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.

BackgroundCranial neural crest cells (NCCs) are a unique embryonic cell type which give rise to a diverse array of derivatives extending from neurons and glia through to bone and cartilage. Depending on their point of origin along the antero-posterior axis cranial NCCs are rapidly sorted into distinct migratory streams that give rise to axial specific structures. These migratory streams mirror the underlying segmentation of the brain with NCCs exiting the diencephalon and midbrain following distinct paths compared to those exiting the hindbrain rhombomeres (r). The genetic landscape of cranial NCCs arising at different axial levels remains unknown.ResultsHere we have used RNA sequencing to uncover the transcriptional profiles of mouse cranial NCCs arising at different axial levels. Whole transcriptome analysis identified over 120 transcripts differentially expressed between NCCs arising anterior to r3 (referred to as r1-r2 migratory stream for simplicity) and the r4 migratory stream. Eight of the genes differentially expressed between these populations were validated by RT-PCR with 2 being further validated by in situ hybridisation. We also explored the expression of the Neuropilins (Nrp1 and Nrp2) and their co-receptors and show that the A-type Plexins are differentially expressed in different cranial NCC streams.ConclusionsOur analyses identify a large number of genes differentially regulated between cranial NCCs arising at different axial levels. This data provides a comprehensive description of the genetic landscape driving diversity of distinct cranial NCC streams and provides novel insight into the regulatory networks controlling the formation of specific skeletal elements and the mechanisms promoting migration along different paths.

BackgroundThe molecular mechanisms underlying the development of the unusual echinoderm pentameral body plan and their likeness to mechanisms underlying the development of the bilateral plans of other deuterostomes are of interest in tracing body plan evolution. In this first study of the spatial expression of genes associated with Nodal and BMP2/4 signalling during the transition to pentamery in sea urchins, we investigate Heliocidaris erythrogramma, a species that provides access to the developing adult rudiment within days of fertilization.ResultsBMP2/4, and the putative downstream genes, Six1/2, Eya, Tbx2/3 and Msx were expressed in the earliest morphological manifestation of pentamery during development, the five hydrocoele lobes. The formation of the vestibular ectoderm, the specialized region overlying the left coelom that forms adult ectoderm, involved the expression of putative Nodal target genes Chordin, Gsc and BMP2/4 and putative BMP2/4 target genes Dlx, Msx and Tbx. The expression of Nodal, Lefty and Pitx2 in the right ectoderm, and Pitx2 in the right coelom, was as previously observed in other sea urchins.ConclusionThat genes associated with Nodal and BMP2/4 signalling are expressed in the hydrocoele lobes, indicates that they have a role in the developmental transition to pentamery, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. We suggest that the Nodal and BMP2/4 signalling cascades might have been duplicated or split during the evolution to pentamery.

BackgroundLeft-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet.ResultsHere we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.ConclusionsBased on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0128-7) contains supplementary material, which is available to authorized users.