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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies

Eyespot patterns of nymphalid butterflies are an example of a novel trait yet, the developmental origin of eyespots is still not well understood. Several genes have been associated with eyespot development but few have been tested for function. One of these genes is the signaling ligand, wingless, which is expressed in the eyespot centers during early pupation and may function in eyespot signaling and color ring differentiation. Here we tested the function of wingless in wing and eyespot development by down-regulating it in transgenic Bicyclus anynana butterflies via RNAi driven by an inducible heat-shock promoter. Heat-shocks applied during larval and early pupal development led to significant decreases in wingless mRNA levels and to decreases in eyespot size and wing size in adult butterflies. We conclude that wingless is a positive regulator of eyespot and wing development in B. anynana butterflies.

developmental biology↗

bioRxiv2017

Insm1a regulates motor neuron development in zebrafish

Insulinoma-associated1a (Insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insmla was a vital regulator of motor neuron development and provide a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insmla in spinal cord and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization and imaging analysis of transgenic reporter line Tg(insmla: mCherry) ntu805. Then we demonstrated that the deficiency of insmla in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP) and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that insmla-null zebrafish significantly reduced the swimming activity. Furthermore, we proved that the insmla loss of function significantly decreased the transcripts levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insmla deficient embryos. Taken together, these data indicate that insmla regulates the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.

developmental biology↗

bioRxiv2017

Hippo signaling restricts cells in the second heart field that differentiate into Islet-1-positive atrial cardiomyocytes

AbstractCardiac precursor cells (CPCs) in the first heart field (FHF) and the second heart field (SHF) present at both arterial and venous poles assemble to form a cardiac tube in zebrafish. Hippo kinase cascade is essential for proper heart formation; however, it remains elusive how Hippo signal contributes to early cardiac fate determination. We here demonstrate that mutants of large tumor suppressor kinase 1/2 (lats1/2) exhibited an increase in a SHF marker, Islet1 (Isl1)-positive and hand2 promoter-activated venous pole atrial cardiomyocytes (CMs) and that those showed expansion of the domain between between the anterior and the posterior lateral plate mesoderm. Consistently, TEAD-8 dependent transcription was activated in caudal region of the left ALPM cells that gave rise to the venous pole atrial CMs. Yap1/Wwtr1-promoted bmp2b expression was essential for Smad-regulated hand2 expression in the left ALPM, indicating that Hippo signaling restricts the SHF cells originating from the left ALPM that move toward the venous pole.

developmental biology↗

bioRxiv2017

Dual functions of Discoidin domain receptor coordinate cell-matrix adhesion and collective polarity in migratory cardiopharyngeal progenitors

Integrated analyses of regulated effector genes, cellular processes, and extrinsic signals are required to understand how transcriptional networks coordinate fate specification and cell behavior during embryogenesis. Migratory pairs of cardiac progenitors in the tunicate Ciona provide the simplest model of collective migration in chordate embryos. Ciona cardiopharyngeal progenitors (aka trunk ventral cells, TVCs) polarize as leader and trailer cells, and migrate between the ventral epidermis and trunk endoderm, which influences collective polarity. Using functional perturbations and quantitative analyses, we show that the TVC-specific and collagen-binding Discoidin-domain receptor (Ddr) cooperates with Integrin-{beta}1 to promote cell-matrix adhesion to the epidermis. We found that endoderm cells secrete a collagen, Col9-a1, that is deposited in the basal epidermal matrix and activates Ddr at the ventral membrane of migrating TVCs. A functional antagonism between Ddr/Int{beta}1-mediated cell-matrix adhesion and Vegfr signaling appears to modulate the position of cardiopharyngeal progenitors between the endoderm and epidermis. Finally, we show that Ddr activity promotes leader-trailer-polarized BMP-Smad signaling independently of its role in cell-matrix adhesion. We propose that dual functions of Ddr act downstream of cardiopharyngeal-specific transcriptional inputs to coordinate subcellular processes underlying collective polarity and directed migration.

developmental biology↗

bioRxiv2017

A zebrafish model of developmental joint dysplasia: Manipulating the larval mechanical environment to drive the malformation and recovery of joint shape

Developmental dysplasia of the hip (DDH), a malformation of the acetabulum, is a frequent cause of early onset osteoarthritis. The disease encompasses a spectrum of severities, some of which are more amenable to treatment. Embryonic immobilisation significantly impairs the development of joint shape however the impact of this malformation to the function and growth of the joint in the short to medium term is unclear. We developed a novel model of developmental joint dysplasia using the zebrafish jaw joint to identify the mechanisms regulating cellular plasticity and ability to recover joint shape and function. Larval zebrafish were immobilised either pharmacologically or using targeted ablation of jaw muscles to induce an altered joint shape. Following restoration of muscle activity we dynamically monitored the joint shape and function in individuals at cellular resolution impossible in other vertebrate species. Reflecting the variability of the human condition we found a proportion of joints will recover both their shape and function, while others will not; despite coming from a genetically homogenous population. This allowed us to study what controls likelihood of recovery; we identified a number of cellular changes that predict likelihood of functional recovery, including position of precursor cells, and specific patterns of proliferation, migration and differentiation in joints and associated connective tissues. These factors together predict recovery better than severity of malformation alone. Using Finite Element Analysis we studied the mechanics of joints representative of ones that recover and those that fail to identify differences in patterns of strain that could explain the cellular behaviours that underpin likelihood of recovery. Thus, this model would enable the study of the short to long term impact of altered joint shape on function and could help to identify the changes that render an individual more receptive to treatment and therefore may potentially be indicative of long term joint health.

developmental biology↗

bioRxiv2017

Xbra and Smad-1 response elements cooperate in PV.1 promoter to inhibit the early neurogenesis in Xenopus embryos

Crosstalk of signaling pathways plays crucial roles in cell fate determination, cell differentiation and proliferation. Both BMP-4/Smad-1 and FGF/Xbra signaling induce the expression of PV.1, leading to neural inhibition. However, BMP-4/Smad-1 and FGF/Xbra signaling crosstalk in the regulation of PV.1 transcription is still largely unknown. In this study, Smad-1 and Xbra physically interacted and regulated the PV.1 transcriptional activation in a synergistic manner. Xbra and Smad-1 directly bound within the proximal region of the PV.1 promoter and cooperatively enhanced the binding of an interacting partner within the promoter. Maximum cooperation was achieved in the presence of intact DNA binding sites of both Smad-1 and Xbra. Collectively, BMP-4/Smad-1 and FGF/Xbra signal crosstalk was required to activate the PV.1 transcription, synergistically. Suggesting that crosstalk of BMP-4 and FGF signaling facilitates the fine-tuning regulation of PV.1 transcription to inhibit neurogenesis during embryonic development of Xenopus.\n\nSummary statementFGF/Xbra positively regulates the PV.1 expression in the Xenopus via an unknown mechanism. Our study shows that both BMP-4/Smad-1 and FGF/Xbra exhibits a signaling crosstalk to regulate PV.1 transcription activation, promoting to ectoderm and mesoderm formation and inhibiting the early neurogenesis in Xenopus.

developmental biology↗

bioRxiv2017

Hes1 oscillations synchronize and refine condensation formation and patterning of the avian limb skeleton

The tetrapod appendicular skeleton is initiated as spatially patterned mesenchymal condensations. The size and spacing of these condensations in avian limb buds are mediated by a reaction-diffusion-adhesion network consisting of galectins Gal-1A, Gal-8 and their cell surface receptors. In cell cultures, the appearance of condensations is synchronized across distances greater than the characteristic wavelength of their spatial pattern. We explored the possible role of observed oscillations of the transcriptional co-regulator Hes1 in this phenomenon. Treatment of micromass cultures with DAPT, a {gamma}-secretase inhibitor, damped Hes1 oscillations, elevated Gal-1A and -8 mRNA levels, and led to irregularly-sized and fused condensations. In developing limb buds, DAPT led to spatially non-uniform Hes1 expression and fused and misshapen digits. Periodicity in adhesive response to Gal-1A, a plausible Hes1-dependent function, was added to a previously tested mathematical model for condensation patterning by the two-galectin network. The enhanced model predicted regularization of patterning due to synchronization of Hes1 oscillations and resulting spatiotemporal coordination of its expression. The model also predicted changes in galectin expression and patterning in response to suppression of Hes1 expression, which were confirmed in in vitro experiments. Our results indicate that the two-galectin patterning network is regulated by Hes1 dynamics, the synchronization of which refines and regularizes limb skeletogenesis.

developmental biology↗

bioRxiv2017

ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

Abbreviations used in this study\n\nAbstractInner cell Mass (ICM) specification into epiblast (Epi) and primitive endoderm (PrE) is an asynchronous and progressive process taking place between E3.0 to E3.75 under the control of the FGF/ERK signaling pathway. Here, we have analyzed in details the kinetics of specification and found that ICM cell responsiveness to the up and down regulation of FGF signaling activity are temporally distinct. We also showed that PrE progenitors are generated later than Epi progenitors. We further demonstrated that, during this late phase of specification, a 4 hours period of FGF/ERK inhibition prior E3.75 is sufficient to convert ICM cells into Epi. Finally, we showed that ICM conversion into Epi in response to inhibition during this short time window requires both transcription and proteasome degradation. Collectively, our data give new insights into the timing and mechanisms involved in the process of ICM specification.

developmental biology↗

bioRxiv2017

Physiological tolerance of the early life history stages of fresh water prawn (Macrobrachium rosenbergii De Man, 1879) to environmental stress

Global climate change is transforming life on earth, causing widespread effects on all ecosystems. Among marine ecosystems, estuaries are considered as nursery grounds for marine and fresh water species. M. rosenbergii, a euryhaline species, migrate to the estuaries for breeding and spawning. The subsequent larval rearing takes place by experiencing variations in temperature and salinity conditions. The present study examines the effect of different temperature and salinity on the larval development and survival by observations on stored yolk utilization, cardiac performance, as well as changes in the rate of growth in body appendages and larval activity. The larvae showed 100% mortality at higher temperature (33.5 {+/-} 0.5 {degrees}C) in all the salinity conditions (12 PPT, 15 PPT, and 20 PPT). The survival rate varied between 76-96 % on exposure to lesser temperature conditions. Likewise, the post-embryonic yolk lasted for 4 days at ambient temperature (29 {degrees}C); whereas, at 33.5 {+/-} 0.5 {degrees}C, it lasted only for 2-3 days. There was an increase in total length of larvae, when exposed to higher temperature and salinity, independently or in combination, but at 33.5 {+/-} 0.5 {degrees}C under all salinity conditions the larvae died on the 5th day. For the cardiac performance, larval heart beat (fH) significantly increased for higher temperature and salinity conditions (20 PPT; 33.5 {degrees}C) and lowered at ambient condition 12 PPT; 29{degrees}C. Larval stroke volume Vs, and Cardiac output Q{square} were higher in ambient conditions and lowest in higher temperature and salinity conditions. However, temperature and salinity together did not show any significant effect on cardiac performance. On the other hand, the larval activity decreased significantly at higher temperature and salinity conditions, compared to ambient conditions but the interactive effect did not show any change. Thus, the physiological responses to temperature and salinity by the early life stages of M. rosenbergii could restrain the tolerance capability of the organism, thereby interfering in the successful completion of the larval development under the altered climatic conditions.

developmental biology↗

bioRxiv2017

Three-dimensional cell neighbourhood impacts differentiation in the inner mass cells of the mouse blastocyst

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterised by the transcription factors NANOG and GATA6, respectively. Here, we present quantitative three-dimensional single cell-based neighbourhood analyses to investigate the spatial distribution of NANOG and GATA6 expression in the ICM of the mouse blastocyst. The cell neighbourhood is characterised by the expression levels of the fate markers in the surrounding cells, together with the number of surrounding cells and cell position. We find that cell neighbourhoods are established in early blastocysts and different for cells expressing different levels of NANOG and GATA6. Highest NANOG expressing cells occupy specific positions within the ICM and are surrounded by 9 neighbours, while GATA6 expressing cells cluster according to their GATA6 levels. The analysis of mutants reveals that NANOG local neighbourhood is regulated by GATA6.\n\nSummary statementThree-dimensional cell neighbourhood, which includes fate marker levels, number of neighbouring cells and cell position, determines cell fate decision in early mouse embryos.

developmental biology↗

bioRxiv2017

In vivo Imaging β-cell Function Reveals Two Waves of β-cell Maturation

The insulin-secreting cells generated from stem cells in vitro are less glucose responsive than primary {beta}-cells. To search for the missing ingredients that are needed for {beta}-cell maturation, we have longitudinally monitored function of every {beta}-cell in Tg (ins:Rcamp1.07) zebrafish embryos with a newly-invented two-photon light-sheet microscope. We have shown that {beta}-cell maturation begins from the islet mantle and propagates to the islet core during the hatching period, coordinated by the islet vascularization. Lower concentration of glucose is optimal to initiate {beta}-cell maturation, while increased glucose delivery to every cell through microcirculation is required for functional boosting of the {beta}-cells. Both the initiation and the boosting of {beta}-cell maturation demands activation of calcineurin/NFAT by glucose. Calcineurin activator combined with glucose promotes mouse neonatal {beta}-cells cultured in vitro to mature to a functional state similar to adult {beta}-cells, suggesting a new strategy for improving stem cell-derived {beta}-like cell function in vitro.

developmental biology↗

bioRxiv2017

Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm muscle causes a reduction in size of the diaphragm central tendon. Replacement of tendon for muscle is likely responsible for the reduced stamina of abl2 mutant mice. Further, ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon in abl2+/- mice. Induction of scleraxis in tendon cells at the ends of these muscle islands suggests that myofibers stimulate tendon differentiation, which in turn regulates myofiber length.

developmental biology↗

bioRxiv2017

Nuclear androgen receptor regulates testes organization and oocyte maturation in zebrafish

Androgens act through the nuclear androgen receptor (AR) to regulate gonad differentiation and development. In mice, AR is required for spermatogenesis, testis development and formation of external genitalia in males and oocyte maturation in females. However, the extent to which these phenotypes are conserved in nonmammalian vertebrates is not well understood, because mutations in AR have not been generated in any other species. Here, we generate zebrafish with a mutation in the ar gene and examine the role of AR on sexual differentiation and gonad development. We find that zebrafish AR is not required for male sexual differentiation, since a portion of ar mutants develop a testis. However, we show that in zebrafish, as in mice, AR is required for the development of secondary sexual characteristics and for proper organization of the testis in males and for oocyte maturation in females. Additionally, we find that zebrafish ar mutant males have functional, mature sperm present in their testis, but are infertile due to an inability to release sperm. These findings suggest that AR is required for male sexual development and fertility, but not essential for sexual differentiation in zebrafish. The ar mutant we developed will be useful for modeling human endocrine function in zebrafish.

developmental biology↗

bioRxiv2017

Cortical area and subcortical volume mediate the effect of parental education and adverse experiences on cognitive performance in youth

Early adversity and socioeconomic disadvantage are risk factors associated with diminished cognitive outcomes during development. Recent studies also provide evidence that upbringings characterized by stressful experiences and markers of disadvantage during childhood, such as lower parental education or household income, are associated with variation in brain structure. Although disadvantage often confers adversity, these are distinct risk factors whose differential influences on neurodevelopment and neurocognitive outcomes are not well characterized. We examined pathways linking parental education, adverse experiences, brain structure, and cognitive performances through an analysis of 1,413 typically-developing youth, ages 8 through 21, in the Philadelphia Neurodevelopmental Cohort. Parental education and adverse experiences had unique associations with cortical surface area and subcortical volume as well as cognitive performance across several domains. Associations between parental education and several cognitive tasks were explained, in part, by variation in cortical surface area. In contrast, associations between adversity and cognitive tasks were explained primarily by variation in subcortical volume. A composite neurodevelopmental factor derived from principal component analysis of cortical thickness, cortical surface area, and subcortical volume mediated independent associations between both parental education and adverse experiences with reading, geometric reasoning, verbal reasoning, attention, and emotional differentiation tasks. Our analysis provides novel evidence that socioeconomic disadvantage and adversity influence neurodevelopmental pathways associated with cognitive outcomes through independent mechanisms.

developmental biology↗

bioRxiv2017

Feedback regulation of steady-state epithelial turnover and organ size

Epithelial organs undergo steady-state turnover throughout adult life, with old cells being continually replaced by the progeny of stem cell divisions1. To avoid hyperplasia or atrophy, organ turnover demands strict equilibration of cell production and loss2-4. However, the mechanistic basis of this equilibrium is unknown. Using the adult Drosophila intestine5, we find that robustly precise turnover arises through a coupling mechanism in which enterocyte apoptosis breaks feedback inhibition of stem cell divisions. Healthy enterocytes inhibit stem cell division through E-cadherin, which prevents secretion of mitogenic EGFs by repressing transcription of the EGF maturation factor rhomboid. Individual apoptotic enterocytes promote divisions by loss of E-cadherin, which releases cadherin-associated {beta}-catenin/Armadillo and p120-catenin to induce rhomboid. Induction of rhomboid in the dying enterocyte triggers EGFR activation in stem cells within a discrete radius. When we block apoptosis, E-cadherin-controlled feedback suppresses divisions, and the organ retains the same number of cells. When we disrupt feedback, apoptosis and divisions are uncoupled, and the organ develops either hyperplasia or atrophy. Altogether, our work demonstrates that robust cellular balance hinges on the obligate coupling of divisions to apoptosis, which limits the proliferative potential of a stem cell to the precise time and place that a replacement cell is needed. In this manner, localized cell-cell communication gives rise to tissue-level homeostatic equilibrium and constant organ size.

developmental biology↗

bioRxiv2017

Cadmium Disrupts Vestibular Function by Interfering with Otolith Formation

Cadmium (Cd2+) is a transition metal found ubiquitously in the earths crust and is extracted in the production of other metals such as copper, lead, and zinc1,2. Human exposure to Cd2+ occurs through food consumption, cigarette smoking, and the combustion of fossil fuels. Cd2+ has been shown to be nephrotoxic, neurotoxic, and osteotoxic, and is a known carcinogen. Animal studies and epidemiological studies have linked prenatal Cd2+ exposure to hyperactivity and balance disorders although the mechanisms remain unknown. In this study we show that zebrafish developmentally exposed to Cd2+ exhibit abnormal otolith development and show an increased tendency to swim in circles, observations that are consistent with an otolith-mediated vestibular defect, in addition to being hyperactive. We also demonstrate that the addition of calcium rescues otolith malformation and reduces circling behavior but has no ameliorating effect on hyperactivity, suggesting that hyperactivity and balance disorders in human populations exposed to Cd are manifestations of separate underlying molecular pathways.

developmental biology↗

bioRxiv2017

Spatial protein analysis in developing tissues: a sampling-based image processing approach

Advances in fluorescence microscopy approaches have made it relatively easy to generate multi-dimensional image volumes and have highlighted the need for flexible image analysis tools for the extraction of quantitative information from such data. Here we demonstrate that by focusing on simplified feature-based nuclear segmentation and probabilistic cytoplasmic detection we can create a tool that is able to extract geometry based information from diverse mammalian tissue images. Our open-source image analysis platform, called SilentMark can cope with noisy images and with crowded fields of cells to quantify signal intensity in different cellular compartments. Additionally, it provides tissue geometry related information, which allows one to quantify protein distribution with respect to marked regions of interest. The lightweight SilentMark algorithms have the advantage of not requiring multiple processors and graphics cards and can be run even with just several hundred MB of memory. This makes it possible to use the method as a web application, effectively eliminating setup hurdles and compatibility issues with operating systems. We test this platform on mouse pre-implantation embryos, embryonic stem cell derived embryoid bodies and mouse embryonic heart and relate protein localisation to tissue geometry.

developmental biology↗

bioRxiv2017

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life yet remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a novel nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

developmental biology↗