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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

Anchor negatively regulates BMP signaling to control Drosophila wing development

Summary statementThe novel gene anchor is the ortholog of vertebrate GPR155, which contributes to preventing wing disc tissue overgrowth and limiting the phosphorylation of Mad in presumptive veins during the pupal stage.\n\nG protein-coupled receptors play a particularly important function in many organisms. The novel Drosophila gene anchor is the ortholog of vertebrate GPR155, and its molecular function and biological process are not yet known, especially in wing development. Knocking down anchor resulted in increased wing size and extra and thickened veins. These abnormal wing phenotypes are similar to those observed in gain-of-function of BMP signaling experiments. We observed that the BMP signaling indicator p-Mad was significantly increased in anchor RNAi-induced wing discs in larvae and that it also abnormally accumulated in intervein regions in pupae. Furthermore, the expression of BMP signaling pathway target genes were examined using a lacZ reporter, and the results indicated that omb and sal were substantially increased in anchor knockdown wing discs. In a study of genetic interactions between Anchor and BMP signaling pathway, the broadened and ectopic vein tissues were rescued by knocking down BMP levels. The results suggested that the function of Anchor is to negatively regulate BMP signaling during wing development and vein formation, and that Anchor targets or works upstream of Dpp.

Developmental Biology↗

bioRxiv2018

Neuropeptides required for Drosophila development under nutritional stress are regulated by the ER-Ca2+ sensor STIM

SignificanceElevation of cytosolic Ca2+ is required for the release of neuropeptides from specialised secretory neurons called neuroendocrine cells. Release of Ca2+ stored in the ER, which further triggers Ca2+ entry from the extracellular milieu is called Store-operated Ca2+ entry (SOCE). STIM, an ER protein, is a key regulator of SOCE and its contribution to neuroendocrine cell functioning is not well studied. Using Drosophila larval development under nutrient restriction as a paradigm, we identified two SOCE regulated neuropeptides, Corazonin and short Neuropeptide F. Reducing STIM alters the level of these neuropeptides in the fed as well as nutrient restricted condition. This study suggests that STIM-triggered SOCE may regulate the release of neuropeptides whose activity ultimately regulates adaptation to nutritional stress.\n\nAbstractCytosolic Ca2+ levels are tightly regulated by the sequestration of Ca2+ within the endoplasmic reticulum (ER). Loss of ER Ca2+ is sensed by STromal Interacting Molecule (STIM), whose translocation to the plasma membrane triggers Store Operated Ca2+ Entry (SOCE), and a subsequent rise in cytosolic Ca2+. Relatively little is known about SOCEs contribution to neuroendocrine cells; a neuronal sub-type that specializes in the secretion of neuropeptides (NPs) which ultimately regulate animal physiology and behavior. To investigate how SOCE regulates NPs, Drosophila development under nutrient restriction (NR) was used as the biological context. Genetic experiments identified the requirement of two SOCE-regulated NPs-corazonin (Crz) and short neuropeptide F (sNPF) - for the development of NR larvae to pupae and finally, adulthood. Overexpression of SOCE regulators was sufficient to rescue the development of NR larvae with reduced sNPF or Crz levels. To facilitate cellular investigations, a restricted set of neurons that produce sNPF and Crz, and are activated by NR, were identified. Immunohistochemistry on these neurons and mass spectrometric measurements of their projections showed that dSTIM regulates Crz and sNPF levels at steady state and in response to NR, likely by modulating their release. Genetically increasing neuronal output, in the background of reduced dSTIM, robustly rescued development of NR larvae. Because NP action fundamentally underpins how animals adapt to stimuli, regulation of NPs by STIM triggered SOCE is likely to be important. In situations where SOCE is altered, such as neurodegenerative diseases, this regulation may contribute to disease manifestation and progression.

developmental biology↗

bioRxiv2018

An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

Control of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive, and reliable procedures to visualise sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. Addition of a cell-permeable protease inhibitor instantly stabilizes newly produced, tagged protein allowing to distinguish recently synthesized protein from more ancient one. After a brief protease inhibitor treatment, the ratio of tagged vs non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged protein it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.

developmental biology↗

bioRxiv2017

Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development

TRIM family members have been implicated in a variety of biological processes such as differentiation and development. We here found that Trim59 plays a critical role in early embryo development from blastocyst stage to gastrula. There existed delayed development and empty yolk sacs from embryonic day (E) 8.5 in Trim59 -/- embryos. No viable Trim59 -/- embryos were observed beyond E9.5. Trim59 deficiency affected primary germ layer formation at the beginning of gastrulation. In Trim59 -/- embryos at E6.5 and E7.5, the expression of primary germ layer formation associated genes including Brachyury, lefty2, Cer1, Otx2, Wnt3 and BMP4 was reduced. Homozygous mutant embryonic epiblast was contracted and the mesoderm was absent. Trim59 could interact with actin and myosin associated proteins. Trim59 deficiency disturbed F-actin polymerization during inner cell mass differentiation. Trim59 mediated polymerization of F-actin was via WASH K63-linked ubiquitination. Thus, Trim59 may be a critical regulator for early embryo development from blastocyst stage to gastrula through modulating F-actin assembly.

developmental biology↗

bioRxiv2026

Male Age and Sexual Maturity: Lipopolysaccharide-induced tumor necrosis factor influences sperm quality and reproduction in Anopheles culicifacies

Elucidating the biological and molecular mechanisms that govern male fertility and mating behavior in mosquitoes is critical for optimizing genetic and sterile insect technique-based vector control strategies. Here, we examined age-related changes in male reproductive capacity in Anopheles culicifacies, using female egg output as an indirect indicator of male fertility. Our results demonstrated that male reproductive age follows a non-linear pattern of fertility. Morphometric analysis from emergence to day 13 post-eclosion revealed a strong correlation between seminal vesicle capacity and female fecundity, suggesting that age-dependent gonadal development directly influences reproductive potential. At the molecular level, we identified AcLITAF6 as a key regulator of male reproductive homeostasis. RNAi-mediated knockdown of AcLITAF6 impaired apoptosis-associated and phagocytic clearance, reduced sperm viability, and decreased female productive outcomes. Conclusively, we reveal a previously unrecognized role of LITAF in sperm quality control and male reproductive fitness, highlighting AcLITAF6 as a potential target for mosquito population suppression strategies.

developmental biology↗

bioRxiv2017

Drosophila beta-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

Summary statementEctotherms thrive within an often remarkable temperature range. At low temperature, betaTub97EF, a beta-tubulin paralog stabilizing microtubules, is upregulated in a tissue-specific manner in the fly Drosophila melanogaster.\n\nAbstractCells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms coping with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different beta-tubulin paralogs, betaTub97EF, was among the genes up-regulated at low temperature in cultured Drosophila cells. As microtubules are known to be cold-sensitive, we analyzed whether betaTub97EF protects microtubules at low temperatures. During development at the optimal temperature (25{degrees}C), betaTub97EF was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14{degrees}C). While betaTub97EF mutants were viable and fertile at 25{degrees}C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing beta-tubulin isoform ratios in hemocytes demonstrated that beta-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, betaTub97EF is required for normal microtubule stability in the gut. These results suggest that betaTub97EF up-regulation at low temperature contributes to acclimation by stabilizing microtubules.

developmental biology↗

bioRxiv2018

Meta-analysis of RNA-seq studies reveals genes responsible for life stage-dominant functions in Schistosoma mansoni

BackgroundSince the genome of the parasitic flatworm Schistosoma mansoni was sequenced in 2009, various RNA-seq studies have been conducted to investigate differential gene expression between certain life stages. Based on these studies, the overview of gene expression in all life stages can improve our understanding of S. mansoni genome biology.\n\nMethodspublicly available RNA-seq data covering all life stages and gonads were mapped to the latest S. mansoni genome. Read counts were normalised across all samples and differential expression analysis was preformed using the generalized linear model (GLM) approach.\n\nResultswe revealed for the first time the dissimilarities among all life stages. Genes that are abundantly-expressed in all life stages, as well as those preferentially-expressed in certain stage(s), were determined. The latter reveals genes responsible for stage-dominant functions of the parasite, which can be a guidance for the investigation and annotation of gene functions. In addition, distinct differential expression patterns were observed between adjacent life stages, which not only correlate well with original individual studies, but also provide additional information on changes in gene expression during parasite transitions. Furthermore, thirteen novel housekeeping genes across all life stages were identified, which is valuable for quantitative studies (e.g., qPCR).\n\nConclusionsthe metaanalysis provides valuable information on the expression and potential functions of S. mansoni genes across all life stages, and can facilitate basic as well as applied research for the community.

developmental biology↗

bioRxiv2017

A Novel Reporter Allele For Monitoring Dll4 Expression Within The Embryonic And Adult Mouse

Canonical Notch signaling requires the presence of a membrane bound ligand and a corresponding transmembrane Notch receptor. Receptor engagement induces multiple proteolytic cleavage events culminating in the nuclear accumulation of the Notch intracellular domain and its binding to a transcriptional co-factor to mediate gene expression. Notch signaling networks are essential regulators of vascular patterning and angiogenesis, as well as myriad other biological processes. Delta-like 4 (Dll4) encodes the earliest Notch ligand detected in arterial cells, and is enriched in sprouting endothelial tip cells. Dll4 expression has often been inferred by proxy using a lacZ knockin reporter allele. This is problematic, as a single copy of Dll4 is haploinsufficient. Additionally, Notch activity regulates Dll4 transcription, making it unclear whether these reporter lines accurately reflect Dll4 expression. Accordingly, accurately defining Dll4 expression is essential for determining its role in development and disease. To address these limitations, we generated a novel BAC transgenic allele with a nuclear-localized {beta}-galactosidase reporter (Dll4-BAC-nlacZ). Through a comparative analysis, we show the BAC line overcomes previous issues of haploinsufficiency, it recapitulates Dll4 expression in vivo, and allows superior visualization and imaging. As such, this novel Dll4 reporter is an important addition to the growing Notch toolkit.\n\nSummary Statement:\n\nWe have developed a novel reporter line, free from complicating factors associated with previous alleles, for monitoring Dll4 expression, at a cellular resolution, in the developing and adult mouse.

developmental biology↗

bioRxiv2018

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

The mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit {beta}-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.\n\nHighlightsO_LIAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.\nC_LIO_LIFzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.\nC_LIO_LIWNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.\nC_LIO_LIEGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.\nC_LI

developmental biology↗

bioRxiv2018

Distinct Sources of a Bovine Blastocyst Digital Image do not Produce the Same Classification by a Previously Trained Software using Artificial Neural Network

We develop an online graphical and intuitive interface connected to a server aiming to facilitate access to professionals worldwide that face problems with bovine blastocysts classification. The interface Blasto3Q (3Q is referred to the three qualities of the blastocyst grading) contains a description of 24 variables that are extracted from the image of the blastocyst and analyzed by three Artificial Neural Networks (ANNs) that classifies the same loaded image. The same embryo (i.e., the biological specimen) was submitted to digital image capture by the control group (inverted microscope with 40x of magnification) and to experimental group (stereomicroscope with maximum of magnification plus 4x zoom from the cell phone). The 36 images obtained from control and experimental groups were uploaded on the Blasto3Q. Each image from both sources was evaluated for segmentation and submitted (only if it could be properly or partially segmented) to the quality grade classification by the three ANNs of the Blasto3Q program. In the group control, all the images were properly segmented, whereas 38.9% (07/18) and 61.1% (11/18) of the images from the experimental group, respectively could not be segmented or were partially segmented. The percentage of agreement was calculated when the same blastocyst was evaluated by the same ANN from the two sources (control and experimental groups). On the 54 potential evaluations of the three ANNs (i.e., 18 images been evaluated by the three networks) from the experimental group only 22.2% agreed with evaluations of the control (12/54). Of the remaining 42 disagreed evaluations from experimental group, 21 were unable to be performed and 21 were wrongly processed when compared with control evaluation.

developmental biology↗

bioRxiv2018

Drosophila small ovary gene ensures germline stem cell maintenance and differentiation by silencing transposons and organising heterochromatin

Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organisation. In our study, we describe a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. We demonstrate that sov is required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. Sov maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed a physical interaction between Sov and HP1a. In the germ cell nuclei, Sov co-localises with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position effect variegation assay, we found a dominant genetic interaction between sov and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. We propose a model in which sov maintains GSC niche integrity by regulating piRNA-mediated transposon silencing as a heterochromatin regulator.\n\nSummary statementSmall ovary maintains the integrity of the stem cell niche by regulating piRNA-mediated transposon silencing acting as a key component of the heterochromatin.

developmental biology↗

bioRxiv2018

Towards identifying subnetworks from FBF binding landscapes in Caenorhabditis spermatogenic or oogenic germlines

Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to \"disentangle\" these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25{degrees}; for comparison, wild-type oogenic hermaphrodites were also raised at 25{degrees}. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.

developmental biology↗

bioRxiv2017

Monsters in the uterus: A parthenogenetic quasi-program causes teratoma-like tumors during aging in wild-type C. elegans

Many diseases whose frequency increases with advancing age are caused by aging (senescence), but the mechanisms of senescence remain poorly understood. According to G.C. Williams and M.V. Blagosklonny, a major etiological determinant of senescence is late-life, wild-type gene action and non-adaptive execution of biological programs (or quasi-programs). These generate a wide range of senescent pathologies causing illness and death. Here we investigate the etiology of a prominent senescent pathology in the nematode C. elegans, uterine tumors, in the light of the Williams Blagosklonny theory. Uterine tumors develop from unfertilized, immature oocytes which execute incomplete embryogenetic programs. This includes extensive endomitosis, leading to formation of chromatin masses and cellular hypertrophy. The starting point of pathogenesis is exhaustion of sperm stocks. The timing of this transition between program and quasi-program can be altered by blocking sperm production (causing earlier tumors) or supplying additional sperm by mating (delaying tumor onset). Other pathophysiological determinants are yolk consumption by tumors, and bacterial proliferation within tumors. Uterine tumors resemble mammalian ovarian teratomas (tera, Greek: monster) in that both develop from oocytes that fail to mature after meiosis I, and both are the result of quasi-programs. Moreover, older but not younger uterine tumors show expression of markers of later embryogenesis, i.e. are teratoma-like. These results show how uterine tumors in C. elegans form as the result of run-on of embryogenetic quasi-programs. They also suggest fundamental etiological equivalence between teratoma and some forms of senescent pathology, insofar as both are caused by quasi-programs.

developmental biology↗

bioRxiv2017

Endophyte Chaetomium globosum D38 and its elicitors promote tanshinones accumulation of Salvia miltiorrhiza

Due to the low yield of tanshinones and their analogues in Salvia miltiorrhiza, there are all kinds of stimulation strategies having been applied to improve tanshinones output in plant tissue cultures. Endophytic fungi have formed various different relationships with their host plants withstanding host and environmental factors, including symbiotic, mutualistic, commensalistic, and parasitic. Thus we take the assumption that endophytic fungi may be an emerging microbial tool used to promote secondary metabolism, which will promote the production of active compounds through endophyte-based biology method. Our study therefore aimed to examine the effects of live endophytic fungus Chaetomium globosum D38 and its elicitors on the accumulation of tanshinones in hairy root cultures of Salvia miltiorrhiza. C. globosum D38 mainly colonized in the intercellular gap of xylem parenchyma cells of S. miltiorrhiza hairy root, during long term co-existence without any toxicity against S. miltiorrhiza hairy root. Moreover, both of the live fungus and its mycelia extracts could induce the production of tanshinones, in special dihydrotanshinone I and cryptotanshinone. The effects of mycelia extracts were much stronger than that of the live fungus on tanshinones synthesis, which increased the transcriptional activity of genes with repect to tanshinone biosynthetic pathway obviously. Our results indicated that both of the live C. globosum D38 and its mycelia extracts could be utilized for tanshinones accumulation in S. miltiorrhiza hairy root. Whats more, D38 also could be made into biotic fertilizer applying into S.miltiorrhiza seddlings, which not only promoted host growth but the tanshinones and phenylpropionic acid accumulation. In the soil environment, D38 had formed bitrophic and mutual beneficial relationship with the host and enhanced the primary metabolism on the whole so as to have facilitative effects on phenylpropionic acid accumulation. To sum up, Chaetomium globosum D38 was a highly effective endophytic fungus for S. miltiorrhiza.

developmental biology↗

bioRxiv2018

A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest.

Multipotent neural crest (NC) progenitors generate an astonishing array of derivatives, including neuronal, skeletal components and pigment cells (chromatophores), but the molecular mechanisms allowing balanced selection of each fate remain unknown. In zebrafish, melanocytes, iridophores and xanthophores, the three chromatophore lineages, are thought to share progenitors and so lend themselves to investigating the complex gene regulatory networks (GRNs) underlying fate segregation of NC progenitors. Although the core GRN governing melanocyte specification has been previously established, those guiding iridophore and xanthophore development remain elusive. Here we focus on the iridophore GRN, where mutant phenotypes identify the transcription factors Sox10, Tfec and Mitfa and the receptor tyrosine kinase, Ltk, as key players. We present expression data, as well as loss and gain of function results, guiding the derivation of an initial iridophore specification GRN. Moreover, we use an iterative process of mathematical modelling, supplemented with a novel, Monte Carlo screening algorithm suited to the qualitative nature of the experimental data, to allow for rigorous predictive exploration of the GRN dynamics. Predictions were experimentally evaluated and testable hypotheses were derived to construct an improved version of the GRN, which we showed produced outputs consistent with experimentally observed gene expression dynamics. Our study reveals multiple important regulatory features, notably a sox10-dependent positive feedback loop between tfec and ltk driving iridophore specification; the molecular basis of sox10 maintenance throughout iridophore development; and the cooperation between sox10 and tfec in driving expression of pnp4a, a key differentiation gene. We also assess a candidate repressor of mitfa, a melanocyte-specific target of sox10. Surprisingly, our data challenge the reported role of Foxd3, an established mitfa repressor, in iridophore regulation. Our study builds upon our previous systems biology approach, by incorporating physiologically-relevant parameter values and rigorous evaluation of parameter values within a qualitative data framework, to establish for the first time the core GRN guiding specification of the iridophore lineage.\n\nAuthor SummaryMultipotent neural crest (NC) progenitors generate an astonishing array of derivatives, including neuronal, skeletal components and pigment cells, but the molecular mechanisms allowing balanced selection of each fate remain unknown. In zebrafish, melanocytes, iridophores and xanthophores, the three chromatophore lineages, are thought to share progenitors and so lend themselves to investigating the complex gene regulatory networks (GRNs) underlying fate segregation of NC progenitors. Although the core GRN governing melanocyte specification has been previously established, those guiding iridophore and xanthophore development remain elusive. Here we present expression data, as well as loss and gain of function results, guiding the derivation of a core iridophore specification GRN. Moreover, we use a process of mathematical modelling and rigorous computational exploration of the GRN to predict gene expression dynamics, assessing them by criteria suited to the qualitative nature of our current understanding of iridophore development. Predictions were experimentally evaluated and testable hypotheses were derived to construct an improved version of the GRN, which we showed produced outputs consistent with experimentally observed gene expression dynamics. The core iridophore GRN defined here is a key stepping stone towards exploring how chromatophores fate decisions are made in multipotent NC progenitors.

developmental biology↗

bioRxiv2017

Genome And Epigenome Engineering CRISPR Toolkit For Probing In Vivo cis-Regulatory Interactions In The Chicken Embryo

CRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present a highly efficient toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise efficient guide-RNA expression from novel chick U6-mini-vectors, provide a strategy for rapid somatic gene knockout and establish protocol for evaluation of mutational penetrance by targeted next generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.\n\nSummary StatementWe present an optimised toolkit for efficient genome and epigenome engineering using CRISPR in chicken embryos, with a particular focus on probing gene regulatory interactions during neural crest development.\n\nList of AbbreviationsGenome Engineering (GE), Epigenome Engineering (EGE), single guide RNA (sgRNA), Neural Crest (NC), Transcription Factor (TF), Next Generation Sequencing (NGS), somite stage (ss), Hamburger Hamilton (HH).

developmental biology↗

bioRxiv2018

A human cell model of cardiac pathophysiological valvulogenesis

Genetically modified mice have advanced our understanding of valve development and related pathologies. Yet, little is known regarding human valvulogenesis in health and diseases. Genuine human in vitro models that reproduce valvular (patho)biology are thus needed. We here developed a human pluripotent stem cell-derived model fit to decode the early steps of human valvulogenesis and to recapitulate valve disease traits in a dish.\n\nUsing cellular based, single cell omics-informed and in vivo-validated approaches, we derived a population of pre-valvular endocardial cells from a pluripotent stem cell source. These human prevalvular cells (HPVCs) expressed gene patterns conforming to the atrio-ventricular canal (AVC) endocardium signature originally established in E9.0 mouse embryos. In fact, HPVC treated with BMP2, cultured onto mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, underwent endothelial-to-mesenchymal transition and expressed markers of valve interstitial cells of different valvular layers demonstrating tissue functionality. HPVCs also differentiated into tendinous/chondrogenic cells in line with the valvular repertoire. Extending this valvulogenic model to patient specific iPS cells, we recapitulated features of mitral valve prolapse and uncovered that dysregulation of the SHH pathway is likely to be at the origin of the disease thus providing a putative therapeutic target.\n\nHuman pluripotent stem cells recapitulate early valvulogenesis and provide a powerful model to systematically decipher the origin and lineage contribution of different valvular cell types in humans as well as to study valve diseases in a dish.

developmental biology↗

bioRxiv2016

Zika Fetal Neuropathogenesis: Etiology of a Viral Syndrome

The ongoing Zika Virus epidemic in the Americas, and the observed association with both fetal abnormalities (primary microcephaly) and adult autoimmune pathology (Guillain-Barre syndrome) has brought attention to this neglected pathogen. While initial case studies generated significant interest in the Zika virus outbreak, larger prospective epidemiology and basic virology studies examining the mechanisms of Zika viral infection and associated pathophysiology are only now starting to be published. In this review, we analyze Zika fetal neuropathogenesis from a comparative pathology perspective, using the historic metaphor of \"TORCH\" viral pathogenesis to provide context. By drawing parallels to other viral infections of the fetus, we identify common themes and mechanisms that may illuminate the observed pathology. The existing data on the susceptibility of various cells to both Zika and other flavivirus infections are summarized. Finally, we highlight relevant aspects of the known molecular mechanisms of flavivirus replication.\n\nKey Learning PointsO_LIViral TORCH pathogens reveal common patterns of fetal pathophysiology and vertical transmission which are relevant to Zika Virus fetal neuropathogenesis.\nC_LIO_LIThe teratogenic effects of Zika Virus infection during the first trimester may involve infection of the trophoblast, viral translocation across the placenta, migration of infected cells resulting in embryonic infection, or indirect effects associated with high levels of inflammatory cytokines produced by infected placenta.\nC_LIO_LIPre-existing maternal non-neutralizing antibody to Zika virus may enhance the probability of infection or more severe disease in the fetus.\nC_LIO_LIAXL has been identified as a major receptor for Zika Virus.\nC_LIO_LIZika virus activation of Toll Like Receptor 3 (TLR-3) pathways in central nervous system cells may trigger apoptosis and attenuate neurogenesis, directly contributing to fetal neuropathology.\nC_LIO_LIFlaviviruses subvert host autophagy and noncoding RNA regulatory pathways.\nC_LIO_LIRecognition of viral sequences by regulatory RNA binding proteins such as Musashi may have a role in Zika pathogenesis and host tissue tropism.\nC_LIO_LIEvidence from other TORCH viral pathogen studies indicate multiple plausible hypotheses for transplacental infection by Zika virus during the second or third trimester, including transcytosis of non-neutralizing antibody-coated Zika virus complexes.\nC_LI\n\nKey ReferencesAdibi JJ, Marques ET Jr, Cartus A, Beigi RH. Teratogenic effects of the Zika virus and the role of the placenta. Lancet 2016; 387: 1587-90 (Hypothesis)\nAdams Waldorf KM, McAdams RM. Influence of infection during pregnancy on fetal development. Reproduction. 2013 Oct 1;146(5) (Review)\nHamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, et al. Biology of Zika Virus Infection in Human Skin Cells. J Virol. 2015;89(17):8880-96.\nMlakar J, Korva M, Tul N, Popovi[c] M, Polj[s]ak-Prijatelj M, Mraz J, et al. Zika Virus Associated with Microcephaly. N Engl J Med. 2016 Feb 10.\nPaul LM, Carlin ER, Jenkins MM, Tan AL, Barcellona CM, Nicholson CO, Trautmann L, Michael SF, Isern S. Dengue Virus Antibodies Enhance Zika Virus Infection. bioRxiv doi: http://dx.doi.org/10.1101/050112\nCrow YJ, Manel N. Aicardi-Goutieres syndrome and the type I interferonopathies. Nat Rev Immunol. 2015;15(7):429-40.\nTonduti D, Orcesi S, Jenkinson EM, Dorboz I, Renaldo F, Panteghini C, et al. Clinical, radiological and possible pathological overlap of cystic leukoencephalopathy without megalencephaly and Aicardi-Goutieres syndrome. Eur J Paediatr Neurol. 2016.\nCipolat Mis MS, Brajkovic S, Frattini E, Di Fonzo A, Corti S. Autophagy in motor neuron disease: Key pathogenetic mechanisms and therapeutic targets. Molecular and Cellular Neurosciences. 2016;72:84-90.\nDang J, Tiwari SK, Lichinchi G, Qin Y, Patil VS, Eroshkin AM, Rana TM. Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3. Cell Stem Cell. 2016: 19: 1-8.\nVianna FS, Schuler-Faccini L, Leite JC, de Sousa SH, da Costa LM, Dias MF, et al. Recognition of the phenotype of thalidomide embryopathy in countries endemic for leprosy: new cases and review of the main dysmorphological findings. Clin Dysmorphol. 2013;22(2):59-63.

Developmental Biology↗