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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Fndc3a (Fibronectin Domain Containing Protein 3A) influences median fin fold development and caudal fin regeneration in zebrafish by ECM alteration.

Summary statementWe investigated potential functions of Fndc3a during caudal fin development and regeneration in zebrafish. Reduced function interferes with correct epidermal cells structure and implies a role during vertebrate extremity development.\n\nAbstractInherited genetic alterations are often found to be disease-causing factors of patient phenotypes. To unravel the molecular consequences of newly identified factors functional investigations in vivo are eminent. We investigated molecular functions of FNDC3A (Fibronectin Domain Containing Protein 3A; HUGO), a novel candidate gene for split-hand/foot malformations (SHFM) in humans, by utilizing zebrafish (Danio rerio) as a vertebrate model. Patients with congenital SHFM display prominent limb malformations, which are caused by disturbance of limb development due to defects in apical ectodermal ridge (AER) establishment and maintenance. Initial gene expression and protein localization studies clarified the presence of fndc3a in developing and regenerating fins of zebrafish. For functional studies we established a hypomorphic fndc3a mutant line (fndc3awue1/wue1) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. Furthermore, fndc3awue1/wue1 mutants display abnormal collagen localization, actinotrichia breakup and cellular defects in epidermal cells during caudal fin development. The observed effects are only temporary and later result in rather normal fin development in adults. In accordance with early fin development, proper caudal fin regeneration in adult fndc3awue1/wue1 mutants is hampered by interference with actinotrichia formation and epidermal cell abnormalities. Investigation of cellular matrix formation implied that loss of ECM structure is a common cause for both phenotypes. Our results thereby provide a molecular link between Fndc3a function during both developmental processes in zebrafish and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development in vertebrates.

developmental biology↗

bioRxiv2018

tudor-domain containing protein 5-prime promotes male sexual identity in the Drosophila germline and is repressed in females by Sex lethal

For sexually reproducing organisms, production of male or female gametes depends on specifying the correct sexual identity in the germline. In D. melanogaster, Sex lethal (Sxl) is the key gene that controls sex determination in both the soma and the germline, but how it does so in the germline is unknown, other than that it is different than in the soma. We conducted an RNA expression profiling experiment to identify direct and indirect germline targets of Sxl specifically in the undifferentiated germline. We find that, in these cells, Sxl loss does not lead to a global masculinization observed at the whole-genome level. In contrast, Sxl appears to affect a discrete set of genes required in the male germline, such as Phf7. We also identify tudor domain containing protein 5-prime (tdrd5p) as a target for Sxl regulation that is important for male germline identity. tdrd5p is repressed by Sxl in female germ cells, but is highly expressed in male germ cells where it promotes proper male fertility and germline differentiation. Additionally, Tdrd5p localizes to cytoplasmic granules with some characteristics of RNA Processing (P-) Bodies, suggesting that it promotes male identity in the germline by regulating post-transcriptional gene expression.\n\nAuthor summaryLike humans, all sexually reproducing organisms require gametes to reproduce. Gametes are made by specialized cells called germ cells, which must have the correct sexual identity information to properly make sperm or eggs. In fruit flies, germ cell sexual identity is controlled by the RNA-binding protein Sxl, which is expressed only in females. To better understand how Sxl promotes female identity, we conducted an RNA expression profiling experiment to identify genes whose expression changes in response to the loss of Sxl from germ cells. Here, we identify tudor domain containing protein 5-prime (tdrd5p), which is expressed 17-fold higher in ovaries lacking Sxl compared to control ovaries. Additionally, tdrd5p plays an important role in males as male flies that are mutant for this gene cannot make sperm properly and thus are less fertile. Moreover, we find that tdrd5p promotes male identity in the germline, as several experiments show that it can shift the germ cell developmental program from female to male. This study tells us that Sxl promotes female identity in germ cells by repressing genes, like tdrd5p, that promote male identity. Future studies into the function of tdrd5p will provide mechanistic insight into how this gene promotes male identity.

developmental biology↗

bioRxiv2018

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Modern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fit-for-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

developmental biology↗

bioRxiv2018

Hydraulic control of embryo size, tissue shape and cell fate

Size control is fundamental in tissue development and homeostasis1,2. While the role of cell proliferation in this process has been widely studied3, the mechanisms of organ size control and how it impacts cell fates remain elusive. Here, we use mouse blastocyst development as a model to unravel a key role of fluid-filled lumen in embryonic size control and cell fate specification. We find that during blastocyst expansion, there is a two-fold increase in the pressure of the lumen that translates into a concomitant increase in the cortical tension of trophectoderm (TE) cells lining the lumen. Increased cortical tension leads to vinculin mechanosensing and maturation of the functional tight junctions, thereby establishing a positive feedback loop to accommodate lumenal growth. However, when the cortical tension reaches a critical threshold, cell-cell adhesion cannot be sustained, and mitotic entry leads to a rupture of TE epithelium, fluid leakage and collapse of the blastocyst cavity. A simple theory of hydraulically-gated oscillations that integrates these feedback interactions recapitulates the evolution of cavity size and predicts the scaling of embryonic size with the tissue volume. Our theory further predicts that reduced cortical tension or disrupted tight junctions, and increased tissue stiffness lead to smaller embryonic size. These predictions are verified experimentally by embryological, pharmacological and genetic manipulations of the embryos. Remarkably, these changes to lumenal size, without a change in the tissue volume, lead to alteration of tissue architecture and cell fate. Overall, our study reveals how lumenal pressure and tissue mechanics control embryonic size at the tissue scale, that in turn couples to cell position and fate at the cellular scale.

developmental biology↗

bioRxiv2018

Maintaining trunk neural crest cells as crestospheres

Neural crest cells have broad migratory and differentiative ability that differs according to their axial level of origin. However, their transient nature has limited understanding of their stem cell and self-renewal properties. While an in vitro culture method has made it possible to maintain cranial neural crest cells as self-renewing multipotent crestospheres (Kerosuo et al., 2015), these same conditions failed to preserve trunk neural crest in a stem-like state. Here we optimize culture conditions for maintenance of trunk crestospheres, comprised of both neural crest stem and progenitor cells. Trunk crestospheres display elevated expression of neural crest cell markers as compared to those characteristic of neural tube or mesodermal fates. Moreover, trunk crestospheres have increased expression of trunk-related markers as compared to cranial genes. Finally, we use lentiviral transduction as a tool to manipulate gene expression in trunk crestospheres. Taken together, this method enables long-term in vitro maintenance and manipulation of trunk neural crest cells in a premigratory stem or early progenitor state to probe the mechanisms underlying their stemness and lineage decisions.\n\nHighlightsO_LITrunk-derived multipotent neural crest stem cells can be cultured as crestospheres\nC_LIO_LITrunk-derived crestospheres require different conditions than cranial\nC_LIO_LITrunk crestospheres consist of neural crest stem and progenitor cells\nC_LIO_LITrunk crestospheres can be efficiently transduced using lentiviral vectors\nC_LI

developmental biology↗

bioRxiv2018

Epigenetic inactivation of miR-203 as a key step in neural crest epithelial-to-mesenchymal transition

miR-203 is a tumor-suppressor microRNA with known functions in cancer metastasis. Here, we explore its normal developmental role in the context of neural crest development. As neural crest cells undergo an epithelial-to-mesenchymal transition to emigrate from the neural tube, miR-203 displays a reciprocal expression pattern with key regulators of neural crest delamination, Phf12 and Snail2, and interacts with their 3UTRs. Ectopic maintenance of miR-203 inhibits neural crest migration, whereas its functional inhibition using a \"sponge\" vector promotes premature neural crest delamination. Bisulfite sequencing further shows that epigenetic repression of miR-203 is mediated by the de novo DNA methyltransferase DNMT3B, whose recruitment to regulatory regions on the miR-203 locus is directed by SNAIL2 in a negative feedback loop. These findings reveal an important role for miR-203 in an epigenetic-microRNA regulatory network that influences the timing of neural crest delamination.\n\nSummary statementThe EMT is a highly conserved process, involving similar levels of regulation in both neural crest and cancer cells. Our work shows an epigenetic-miRNA-gene regulatory circuit, conserved in cancer, which controls the timing of neural crest EMT as well.

developmental biology↗

bioRxiv2018

The Warburg effect and lactate signaling augment Fgf signaling to promote sensory-neural development in the otic vesicle

Recent studies indicate that many developing tissues modify glycolysis to favor lactate synthesis, but how this promotes development is unclear. Using forward and reverse genetics in zebrafish, we show that disrupting the glycolytic gene phosphoglycerate kinase-1 (pgk1) impairs Fgf-dependent development of hair cells and neurons in the otic vesicle and other neurons in the CNS/PNS. Focusing on the otic vesicle, we found that Fgf signaling underperforms in pgk1-/- mutants even when Fgf is transiently overexpressed. Wild-type embryos treated with drugs that block synthesis or secretion of lactate mimic the pgk1-/- phenotype, whereas pgk1-/- mutants are rescued by treatment with exogenous lactate. Lactate treatment of wild-type embryos elevates expression of Etv5b/Erm even when Fgf signaling is blocked. Thus, by raising steady-state levels of Etv5b (a critical effector of the Fgf pathway), lactate renders cells more responsive to dynamic changes in Fgf signaling required by many developing tissues.

developmental biology↗

bioRxiv2018

Nell2 regulates the contralateral-versus-ipsilateral visual projection as a layer-specific positional cue

SUMMARY STATEMENTNell2 is an ipsilateral layer-specific axon guidance cue in the visual thalamus and contributes to establishment of the eye-specific retinogeniculate projection by specifically inhibiting contralateral retinal axons.\n\nABSTRACTIn mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific layers in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN). Although layer-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specificretinogeniculate projection, the molecular nature of such cues has remained elusive. Here we show that the extracellular glycoprotein Nell2 (also known as Nel) is expressed in the dorsomedial region of the dLGN, which corresponds to the layer receiving ipsilateral RGC axons. In Nell2 mutant mice, contralateral RGC axons invaded the ipsilateral layer of the dLGN, and ipsilateral axons terminated in partially fragmented patches, forming a mosaic pattern of contralateral and ipsilateral axon termination zones. In vitro, Nell2 exerted inhibitory effects on contralateral, but not ipsilateral, RGC axons. These results provide evidence that Nell2 acts as a layer-specific positional label in the dLGN that discriminates contralateral and ipsilateral RGC axons, and that it plays essential roles in establishment of the eye-specific projection patterns in the retinogeniculate system.

developmental biology↗

bioRxiv2018

Delayed aneuploidy stress response of neural stem cells impairs adult lifespan in flies

SummaryStudying aneuploidy during organism development has strong limitations, as chronic mitotic perturbations used to generate aneuploidy result in lethality. We developed a genetic tool to induce aneuploidy in an acute and time controlled manner during Drosophila development. This is achieved by reversible depletion of cohesin, a key molecule controlling mitotic fidelity.\n\nLarvae challenged with aneuploidy hatch into adults with severe motor defects shortening their lifespan. Neural stem cells, despite being aneuploid, display a delayed stress response and continue proliferating, resulting in the rapid appearance of chromosomal instability, complex array of karyotypes and cellular abnormalities. Notably, when other brain cell-lineages are forced to self-renew, aneuploidy-associated stress response is significantly delayed, indicating that stemness state confers resistance to aneuploidy. Sparing solely the developing brain from induced aneuploidy is sufficient to rescue motor defects and adult lifespan, suggesting that neural tissue is the most ill-equipped to deal with developmental aneuploidy.\n\nHighlightsO_LIReversible depletion of cohesin results in just a round or two of aberrant cell divisions, generating high levels of aneuploidy.\nC_LIO_LILarvae challenged with aneuploidy during development hatch into impaired adults.\nC_LIO_LIFew cell cycles are sufficient for chromosomal instability to emerge from a previously stable aneuploid state.\nC_LIO_LINeural stemness delays aneuploidy stress response.\nC_LIO_LIProtecting only the neural tissue from aneuploidy rescues adult abnormalities and lifespan.\nC_LI

developmental biology↗

bioRxiv2018

Dual role of Bnl/Fgf signaling in proliferation and endoreplication of Drosophila trachealadult progenitor cells

AbstractAdult progenitor cells activation is a key event in the formation of adult organs during development. The initiation of proliferation of these progenitor cells requires specific temporal signals, mostly of them still unknown. In Drosophila, formation of adult tracheal system depends on the activation of tracheal adult progenitors (tracheoblasts) of Tr4 and Tr5 tracheal metamers specific spiracular branches (SB) during the last larval stage. The mitotic activity of these tracheoblasts generate a pool of tracheal differentiated cells that migrate during pupal development along the larval trachea by the activation of the Branchless (Bnl)/Fibroblast growth factor (FGF) signaling to form the abdominal adult tracheal system. In here, we found that, in addition to migration, Bnl/FGF signaling, mediated by the transcription factor Pointed, is also required for adult progenitor cell proliferation in the SBs. Moreover, we found that tracheoblast proliferation in Tr4 and Tr5 SBs relies on the specific expression of the FGF ligand Bnl in their nearby transverse connective branches. Finally, we also show that, in absence of the transcription factor Cut (Ct), Bnl/FGF signaling induces endoreplication of differentiated tracheoblast daughter cells by in part promoting Fizzy-related (Fzr) expression. Altogether, our results suggest a dual role of Bnl/FGF signaling in tracheal adult progenitors, inducing both proliferation and endoreplication of tracheoblasts in late larval development, depending on the presence or absence of the transcription factor ct, respectively.\n\nAuthor summaryThe generation of adult organs and tissue renewal are complex processes that depend on the proliferation and posterior differentiation of undifferentiated progenitor cells in a temporal coordinated manner. Although many signals that regulate the activity of progenitor cells have been identified, the characterization of the mechanisms underlying the temporal and spatial control of such events remain unknown. The tracheal system of Drosophila, the respiratory organ, forms during embryogenesis and it is remodeled during metamorphosis from quiescent adult progenitor cells that proliferate. We have discovered that this proliferation depends on the activation of the FGF signaling as mutations that either inactivate or over-activate the pathway blocks cell division or induced over-proliferation of progenitor cells, respectively. Interestingly, we have found that the same signaling pathway also controls tracheal progenitor cells differentiation by promoting endoreplication. We found that this dual role of FGF signaling in adult progenitor cells, depends on the presence or absence of the transcription factor Cut. Altogether, our results, reveal the mechanism that control the division and differentiation of progenitor cells and open the possibility that analogous signaling pathway may play a similar role in vertebrate stem cell regulation and tumor growth.

developmental biology↗

bioRxiv2018

Myocardial Notch-Rbpj deletion does not affect heart development or function

During vertebrate cardiac development NOTCH signaling activity in the endocardium is essential for the crosstalk between endocardium and myocardium that initiates ventricular trabeculation and valve primordium formation. This crosstalk leads later to the maturation and compaction of the ventricular chambers and the morphogenesis of the cardiac valves, and its alteration may lead to disease. Although endocardial NOTCH signaling has been shown to be crucial for heart development, its physiological role in the myocardium has not been clearly established. Here we have used a genetic strategy to evaluate the role of NOTCH in myocardial development. We have inactivated the unique and ubiquitous NOTCH effector RBPJ in the early cardiomyocytes progenitors, and examined its consequences in cardiac development and function. Our results demonstrate that mice with cTnT-Cre-mediated myocardial-specific deletion of Rbpj develop to term, with homozygous mutant animals showing normal expression of cardiac development markers, and normal adult heart function. Similar observations have been obtained after Notch1 deletion with cTnT-Cre. We have also deleted Rbpj in both myocardial and endocardial progenitor cells, using the Nkx2.5-Cre driver, resulting in ventricular septal defect (VSD), double outlet right ventricle (DORV), and bicuspid aortic valve (BAV), due to NOTCH signaling abrogation in the endocardium of cardiac valves territories. Our data demonstrate that NOTCH-RBPJ inactivation in the myocardium does not affect heart development or adult cardiac function.

developmental biology↗

bioRxiv2018

Determinants of Minimum Acceptable Diet among 6-23 Months Age Children in Ethiopia: A Multilevel Analysis of The Ethiopian Demographic Health Survey

BackgroundThough infant and young children should be fed according to a minimum acceptable diet to ensure appropriate growth and development, only 7% of Ethiopian 6-23 months age children meet the minimum acceptable dietary standards, which is lower than the national target of 11% set for 2016. Therefore, this study aims to assess the individual and community level determinants of minimum acceptable diet among 6-23 months age children in Ethiopia.\n\nMethodsThis study analyzed retrospectively a cross-sectional data on a weighted sample of 2919 children aged 6-23 months nested within 617 clusters after extracting from Ethiopian Demographic and Health Survey 2016 via the link www.measuredhs.com. By employing bi-variate multilevel logistic regression model, variables which were significant at the p-value < 25 were included in multivariable multilevel logistic regression analysis. Finally, variables with p-value < 0.05 were considered as significant predictors of minimum acceptable diet.\n\nResultsOnly 6.1% of 6-23 months age children feed minimum acceptable diet in Ethiopia. Children 18-23 months age (AOR=3.7, 95%CI 1.9, 7.2), fathers with secondary or higher education (AOR=2.1, 95%CI 1.2, 3.6), Employed mothers (AOR=1.7, 95%CI 1.2, 2.5), mothers have access to drinking water (AOR=1.9, 95%CI 1.2, 2.9), mothers with media exposure (AOR=2.1 95%CI 1.1, 2.7) were positive individual level predictors. Urban mothers (AOR=4.8, 95%CI 1.7, 13.2)) and agrarian dominant region (AOR=5.6, 95%CI 2.2, 14.5) were community level factors that significantly associated with minimum acceptable diet of 6-23 months age children.\n\nConclusionBoth individual and community level factors were significantly associated with minimum acceptable diet of 6-23 months age children in Ethiopia, suggesting that nutritional interventions designed to improve child health should not only be implemented at the individual level but tailored to community context as well.

developmental biology↗

bioRxiv2018

Morphogenesis of neurons and glia within an epithelium

To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined the C. elegans amphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM. Here, we show that amphid neurons and glia exhibit epithelial properties, including tight junctions and apical-basal polarity, and develop in a manner resembling other epithelia. We find that DYF-7 is a fibril-forming apical ECM component that prevents rupture of the tube-shaped glial channel, reminiscent of roles for apical ECM in other narrow epithelial tubes. We also identify a role for FRM-2, a homolog of EPBL15/moe/Yurt which promote epithelial integrity in other systems. Finally, we show that other environmentally-exposed neurons share a requirement for DYF-7. Together, our results suggest that these neurons and glia can be viewed as part of an epithelium continuous with the skin, and are shaped by mechanisms shared with other epithelia.

developmental biology↗

bioRxiv2018

De novo genesis of retinal ganglion cells by targeted expression of KLF4 in vivo

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, innovative therapeutic strategies are needed to protect and replace these projection neurons. It has been shown that endogenous glial cells of the retina, Muller cells, can be directly reprogrammed into late-born retinal interneurons. However, since RGCs are the first neurons born during development, the replacement of damaged RGCs requires the reprograming to an early neurogenic state. Here, we demonstrate that the pluripotency regulator Klf4 is sufficient to reprogram the potency of lineage-restricted retinal progenitor cells (RPCs) to generate RGCs in vivo. Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2. In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo reprogramming. Finally, we demonstrate that Klf4 converts Muller cells into neurons that express markers of RGCs. Altogether, we identified Klf4 as a promising tool to reprogram retinal cells and regenerate RGCs in the mature retina.\n\nSignificance StatementCell fate determination is a key process for development, regeneration and for the design of therapeutic strategies that involve cellular reprogramming. This work shows that the manipulation of a single pluripotency regulator (Klf4) is sufficient to reprogram restricted progenitor cells in vivo. These reprogrammed progenitors reacquire the potency to generate retinal ganglion cells. Ganglion cell degeneration is the leading cause of irreversible blindness; therefore, manipulation of ganglion cell competence is of relevance for human health. Our findings point to Klf4 as a promising tool to develop therapeutic strategies for the replacement of damaged ganglion cells.

developmental biology↗

bioRxiv2018

Bacterial community dynamics during embryonic and larval development of three confamilial echinoids

Development of some animals is influenced by and, in some cases, dependent on the associated microbiota. The timing of when associated bacterial communities are established during the development of marine invertebrates and their subsequent dynamics across stages are known for only a few species. Here, we compare the bacterial communities of three confamilial echinoids from egg to juvenile using sequence-based approaches. Bacterial communities are established on unfertilized eggs and change gradually during embryonic and larval development. Despite the differences amongst these pre-metamorphic stages, approximately thirty-percent of OTUs identified in association with unfertilized egg were present in the juveniles. During embryonic development, host-associated communities diverged from the environmental microbiota but later converged following the onset of larval feeding. Taken together, the data presented here support the hypothesis that bacterial communities are established prior to fertilization and community composition shifts gradually thereafter, all while remaining distinct from the environment. Future work will need to determine the relative influence of the host and bacteria-bacteria interactions in shaping the associated bacterial community to determine the potential functional importance of bacteria during the development of larval sea urchins and benthic marine invertebrates more broadly.

developmental biology↗

bioRxiv2018

SOL1 and SOL2 Regulate Fate Transition and Cell Divisions in the Arabidopsis Stomatal Lineage

In the stomatal lineage, cells make fate transitions from asymmetrically dividing and self-renewing meristemoids, to commitment to the guard mother cell identity, and finally though a single division to create mature, post-mitotic stomatal guard cells. Flexibility in the stomatal lineage allows plants to alter leaf size and stomatal density in response to environmental conditions; however, transitions must be clean and unidirectional in order to produce functional and correctly patterned stomata. Among direct transcriptional targets of the stomatal initiating factor, SPEECHLESS, we found a pair of genes, SOL1 and SOL2, required for effective transitions in the lineage. Here we show that these two genes, which are homologues of the LIN54 DNA-binding components of the mammalian DREAM complex, are expressed in a cell cycle dependent manner and regulate cell fate and division properties in the self-renewing early lineage. In the terminal division of the stomatal lineage, however, these two proteins appear to act in opposition to their closest paralogue, TSO1, revealing complexity in the gene family may enable customization of cell divisions in coordination with development.

developmental biology↗

bioRxiv2018

Developmental Effects of the Pesticide Imidacloprid on Zebrafish Body Length and Mortality

The pesticide imidacloprid, a Neonicotinoid, is widely used and commercially available. Neonicotinoids are antagonists at nicotinic acetylcholine receptors, which can cause neurobehavioral effects in developing organisms. While the effects of other insecticides such as Malathion and Fipronil on zebrafish Danio rerio development have been studied, few studies describe the morphological effects of imidacloprid during zebrafish development. To test the hypothesis that imidacloprid concentration positively correlates to increased mortality and decreased body length, we exposed zebrafish to imidacloprid for five days post fertilization. Body length and embryo mortality were recorded at 1, 2, 3, 4, and 5 days post fertilization in 0 ug/L, 100 ug/L, 1,000 ug/L, and 10,000 ug/L imidacloprid concentrations. These concentrations were chosen to mimic levels that could be reached in the environment especially soon after application. Our results demonstrate statistically increased embryo mortality, with a f crit value of 3.193 and a f value of 3.098 from an ANOVA test, and impaired body length with a dose dependent correlation to the concentration of imidacloprid and a P value of less than 0.01 from a T test. Given the pesticides high prevalence, future studies should be considered to determine if this effect may impact livestock and human development.

developmental biology↗

bioRxiv2018

MicroRNA regulation of BMP signaling; cross-talk between endothelium and vascular smooth muscle cells

Vascular smooth muscle cells (vSMC) are essential to the integrity of blood vessels, and therefore an attractive target of therapeutics aimed at improving vascular function. Smooth muscle cells are one of the few cell types that maintain plasticity and can switch phenotypes from differentiated (contractile) to de-differentiated (synthetic) and vice versa. As small regulatory transcripts, miRNAs act as genetic fine tuners of posttranscriptional events and can act as genetic switches promoting phenotypic switching. The microRNA miR26a targets the BMP signalling effector, smad1. We show that loss of miR26 leads to hemorrhage (a loss of vascular stability) in vivo, suggesting altered vascular differentiation. Reduction in miR26a levels increases smad1 mRNA and phospho-Smad1 (pSmad1) levels. We show that increasing BMP signalling by overexpression of smad1 also leads to hemorrhage and that normalization of Smad1 levels through double knockdown of miR26 and smad1 rescues hemorrhage suggesting a direct relationship between miR26 and smad1 and vascular stability. Using a BMP genetic reporter and pSmad1 staining we show that the effect of miR26 on vascular instability is non-autonomous; BMP signalling is active in embryonic endothelial cells, but not in smooth muscle cells. Nonetheless, increased BMP signalling due to loss of miR26 results in an increase in acta2-expressing smooth muscle cell numbers and promotes a differentiated smooth muscle morphology. Taken together our data suggests that miR26 modulates BMP signalling in endothelial cells and indirectly promotes a differentiated smooth muscle phenotype. Our data also suggests that crosstalk from BMP-responsive endothelium to smooth muscle is important for its differentiation.

developmental biology↗