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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

A common molecular logic determines embryonic stem cell self-renewal and reprogramming

During differentiation and reprogramming new cell identities are generated by reconfiguration of gene regulatory networks. Here we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naive pluripotency. We find that a Boolean network architecture defined for maintenance of naive state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single cell resolution. Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 136 predictions formulated by the dynamic network, finding a predictive accuracy of 78.7%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naive pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.

developmental biology↗

bioRxiv2017

Ancestral perinatal obesogen exposure results in a transgenerational thrifty phenotype in mice

Ancestral environmental exposures to non-mutagenic agents can exert effects in unexposed descendants. This transgenerational inheritance has significant implications for understanding disease etiology. The obesogen hypothesis proposes that exposure to obesogenic chemicals can lead to increased adiposity, in vivo. Here we show that exposure of F0 mice to the obesogen tributyltin (TBT) throughout pregnancy and lactation predisposes unexposed F4 male descendants to obesity when dietary fat is increased. Analyses of body fat, plasma hormone levels, and visceral white adipose tissue DNA methylome and transcriptome collectively indicate that the F4 obesity is consistent with a leptin resistant, \"thrifty phenotype\". Ancestral TBT exposure induces global changes in DNA methylation together with altered expression of metabolism-relevant genes when the F4 animals were exposed to dietary challenges. Analysis of chromatin accessibility in F3 and F4 sperm reveal significant differences between control and TBT groups and significant similarities between F3 and F4 TBT groups that overlap with areas of differential methylation in F4 adipose tissue. Taken together, our data suggest that ancestral TBT exposure induces changes in higher order chromatin organization transmissible through meiosis and mitosis.\n\nNon-technical summaryAncestral obesogen exposure in mice causes obesity in untreated F4 male descendants by inducing heritable changes in genome architecture that predispose these animals to become obese when dietary fat is increased modestly. This result is consistent with these animals having a leptin-resistant, \"thrifty\" phenotype

developmental biology↗

bioRxiv2017

The Drosophila Eukaryotic Initiation Factor eIF6 affects development by regulating apoptosis via the ecdysone pathway

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.

developmental biology↗

bioRxiv2017

Angiogenesis inhibiting capacity of Basella rubra and Syszygium cumini fruit extracts using chorioallantoic membrane assay

Angiogenesis is a physiological process of new blood vessel development from pre-existing capillaries. This process is also the main qualification for tumor growth and plays a vital role in tumor invasion and metastasis. Nevertheless, angiogenesis inhibitors can be used to impede abnormal blood vessel growth. The study was conducted to assess angiogenesis inhibiting potential of B. rubra (Alugbati) and S. cumini (Lumboy) fruit extracts as cell mass growth retardants using Chorioallantoic Membrane (CAM) Assay in Anas platyrhyncho (common Duck) embryo. The treatments were individually compared to Retinol palmitate as positive control and 0.9% sterilized normal saline solution as negative control. Data were gathered and analyzed using Analysis of Variance (ANOVA) and Least Significant Difference (LSD) to test for the significant anti-angiogenic activity and pair-wise comparison among the treatments respectively using a p-value of less than 0.01. Analysis showed that there is no significant difference between the fruit extracts and positive control while a significant difference between the fruit extracts and negative control was observed. Both treatments showed very good anti-angiogenic effect with average scores of 1.33 and 1.67 respectively compared to the positive control with a scoring averaging of 1.78. Furthermore, toxicity test is recommended for both treatments.

developmental biology↗

bioRxiv2017

Morphogen dynamics control patterning in a stem cell model of the human embryo

During embryonic development, diffusible signaling molecules called morphogens are thought to determine cell fates in a concentration-dependent manner1-4, and protocols for directed stem cell differentiation are based on this picture5-8. However, in the mammalian embryo, morphogen concentrations change rapidly compared to the time for making cell fate decisions9-12. It is unknown how changing ligand levels are interpreted, and whether the precise timecourse of ligand exposure plays a role in cell fate decisions. Nodal and BMP4 are morphogens crucial for gastrulation in vertebrates13. Each pathway has distinct receptor complexes that phosphorylate specific signal transducers, known as receptor-Smads, which then complex with the shared cofactor Smad4 to activate target genes14. Here we show in human embryonic stem cells (hESCs) that the response to BMP4 signaling indeed is determined by the ligand concentration, but that unexpectedly, the expression of many mesodermal targets of Activin/Nodal depends on rate of concentration increase. In addition, we use live imaging of hESCs with GFP integrated at the endogenous SMAD4 locus to show that a stem cell model for the human embryo15 generates a wave of Nodal signaling. Cells experience rapidly increasing Nodal specifically in the region of mesendoderm differentiation. We also demonstrate that pulsatile stimulation with Activin induces repeated strong signaling and enhances mesoderm differentiation. Our results break with the paradigm of concentration-dependent differentiation and demonstrate an important role for morphogen dynamics in the cell fate decisions associated with mammalian gastrulation. They suggest a highly dynamic picture of embryonic patterning where some cell fates depend on rapid concentration increase rather than absolute levels, and point to ligand dynamics as a new dimension to optimize protocols for directed stem cell differentiation.

developmental biology↗

bioRxiv2017

Proteolytic control of centrosome activity by APC/C-Polo maintains oocyte fate in Drosophila

A universal feature of metazoan reproduction is the elimination of the maternal centrosomes prior to the end of oogenesis. In animals that have a syncytial cyst stage of oocyte development, including Drosophila and mouse, the germline centrosomes undergo a migration to all reside within the oocyte. However, the functional significance of centrosome transport within the female germline and the mechanism orchestrating this event are still a mystery. The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit ubiquitin ligase (E3) that temporally regulates progression of the cell cycle as well as the centrosome cycle. By altering the negative regulation of the cooperating ubiquitin conjugating enzyme (E2), Vihar/Ube2c, we show that temporal control of APC/C activity ensures centrosome stability and migration during early Drosophila oogenesis. When there is perduring APC/C activity, Polo kinase is precociously targeted for destruction, which results in centriole instability and decreased centrosome transport to the oocyte. We show that decreased centrosome transport correlates with a decreased accumulation of pericentriolar material (PCM) proteins on the oocyte nucleus, which results in a weakening of the structural integrity of the egg chamber and loss of oocyte fate - the overall consequence being a reduction in female fertility. Considering the conserved roles of the APC/C and Polo kinase throughout the animal kingdom and the fact that many animals have a syncytial stage of egg development, our results provide insight into the general necessity of gametic centrosome transport for female fertility.

developmental biology↗

bioRxiv2017

Four new induced pluripotent stem cell lines produced from northern white rhinoceros with non-integrating reprogramming factors

Since we first published methods for generation of induced pluripotent stem cells from endangered species1, we have developed improved non-integrating methods for reprogramming the functionally extinct northern white rhinoceros, and generated iPSCs from four more individuals. Our work is part of a long-term plan for assisted reproduction for conservation of endangered species.

developmental biology↗

bioRxiv2017

C. elegans Multidrug Resistance Protein 5 (MRP-5) Transports Vitamin B12 from the Intestine to the Gonad to Support Embryonic Development

Vitamin B12 functions as a cofactor for methionine synthase to produce the anabolic methyl donor S-adenosylmethionine (SAM) and for methylmalonyl-CoA mutase to catabolize the short chain fatty acid propionate. In the nematode Caenorhabditis elegans, maternally supplied vitamin B12 is required for the development of her offspring. However, the mechanism for exporting vitamin B12 from the mother to her offspring is not yet known. Here, we use RNAi of more than 200 transporters with a vitamin B12-sensor transgene to identify the ABC transporter MRP-5 as a candidate vitamin B12 exporter. We show that injection of vitamin B12 into the gonad of mrp-5 deficient mothers rescues embryonic lethality in her offspring. Altogether, our findings identify a maternal mechanism for the transit of an essential vitamin to support the development of the next generation.

developmental biology↗

bioRxiv2017

Jak-Stat pathway induces Drosophila follicle elongation by a gradient of apical contractility

Tissue elongation and its control by spatiotemporal signals is a major developmental question. Currently, it is thought that Drosophila ovarian follicular epithelium elongation requires the planar polarization of the basal domain cytoskeleton and of the extra-cellular matrix, associated with a dynamic process of rotation around the anteroposterior axis. Here we show, by careful kinetic analysis of fat2 mutants, that neither basal planar polarization nor rotation is required during a first phase of follicle elongation. Conversely, a JAK-STAT signaling gradient from each follicle pole orients early elongation. JAK-STAT controls apical pulsatile contractions, and Myosin II activity inhibition affects both pulses and early elongation. Early elongation is associated with apical constriction at the poles and oriented cell rearrangements, but without any visible planar cell polarization of the apical domain. Thus, a morphogen gradient can trigger tissue elongation via a control of cell pulsing and without planar cell polarity requirement.\n\nImpact StatementFollicle elongation does not rely solely on the basal side of the cells but also requires a mechanism integrating a developmental cue with a morphogenetic process involving their apical domain.

developmental biology↗

bioRxiv2017

Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain by scGESTALT

Hundreds of cell types are generated during development, but their lineage relationships are largely elusive. Here we report a technology, scGESTALT, which combines cell type identification by single-cell RNA sequencing with lineage recording by cumulative barcode editing. We sequenced ~60,000 transcriptomes from the juvenile zebrafish brain and identified more than 100 cell types and marker genes. We engineered an inducible system that combines early and late barcode editing and isolated thousands of single-cell transcriptomes and their associated barcodes. The large diversity of edited barcodes and cell types enabled the generation of lineage trees with hundreds of branches. Inspection of lineage trajectories identified restrictions at the level of cell types and brain regions and helped uncover gene expression cascades during differentiation. These results establish scGESTALT as a new and widely applicable tool to simultaneously characterize the molecular identities and lineage histories of thousands of cells during development and disease.

developmental biology↗

bioRxiv2017

Cooperative recruitment of Yan to paired high affinity ETS sites organizes repression to confer specificity and robustness to cardiac cell fate specification

Cis regulatory elements (CREs) are defined by unique combinations of transcription factor binding sites. Emerging evidence suggests that the number, affinity and organization of sites play important roles in regulating enhancer output and ultimately gene expression. Here, we investigate how the cis-regulatory logic of a tissue-specific CRE responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two ETS members, the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, we show that Yan and Pnt have distinct preferences for affinity of sites. Not only does Yan prefer high affinity sites, but a tandem pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and to block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the cooperative Yan recruitment promoted by this conserved high affinity ETS pair not only biases Yan-Pnt competition at the specific CRE, but also organizes Yan repressive complexes in 3D across the eve locus. Taken together our results uncover a novel mechanism by which differential interpretation of CRE syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions.

developmental biology↗

bioRxiv2017

Two distinct mechanisms silence chinmo in Drosophila neuroblasts and neuroepithelial cells to limit their self-renewal

Whether common principles regulate the self-renewing potential of neural stem cells (NSCs) throughout the developing central nervous system is still unclear. In the Drosophila ventral nerve cord and central brain, asymmetrically dividing NSCs, called neuroblasts (NBs), progress through a series of sequentially expressed transcription factors that limits self-renewal by silencing a genetic module involving the transcription factor Chinmo. Here, we find that Chinmo also promotes neuroepithelium growth in the optic lobe during early larval stages by boosting symmetric self-renewing divisions while preventing differentiation. Neuroepithelium differentiation in late larvae requires the transcriptional silencing of chinmo by ecdysone, the main steroid hormone, therefore allowing coordination of NSC self-renewal with organismal growth. In contrast, chinmo silencing in NBs is post-transcriptional and does not require ecdysone. Thus, during Drosophila development, humoral cues or tissue-intrinsic temporal specification programs respectively limit self-renewal in different types of neural progenitors through the transcriptional and post-transcriptional regulation of the same transcription factor.\n\nSUMMARY STATEMENTHere, we demonstrate that the transcription factor chinmo acts as a master gene of NSC self-renewal in the different regions of the developing Drosophila brain where it is controlled by distinct regulatory strategies.

developmental biology↗

bioRxiv2017

spe-43 is required for sperm activation in C. elegans

Successful fertilization requires that sperm are activated prior to contacting an oocyte. In C. elegans, this activation process, called spermiogenesis, transforms round immobile spermatids into motile, fertilization-competent spermatozoa. We describe the phenotypic and genetic characterization of spe-43, a new component of the spe-8 pathway, which is required for spermiogenesis in hermaphrodites; spe-43 hermaphrodites are self-sterile, while spe-43 males show wild-type fertility. When exposed to Pronase to activate sperm in vitro, spe-43 spermatids form long rigid spikes radiating outward from the cell periphery instead of forming a motile pseudopod, indicating that spermiogenesis initiates but is not completed. Using a combination of recombinant and deletion mapping and whole genome sequencing, we identified F09E8.1 as spe-43. SPE-43 is predicted to exist in two isoforms; one isoform appears to be a single-pass transmembrane protein while the other is predicted to be a secreted protein. SPE-43 can bind to other known sperm proteins, including SPE-4 and SPE-29, which are known to impact spermiogenesis. In summary, we have identified a membrane protein that is present in C. elegans sperm and is required for sperm activation via the hermaphrodite activation signal.

developmental biology↗

bioRxiv2017

Coordinated morphogenesis through tension-induced planar polarity

Tissues from different developmental origins must interact to achieve coordinated morphogenesis at the level of a whole organism. C. elegans embryonic elongation is controlled by actomyosin dynamics which trigger cell shape changes in the epidermis and by muscle contractions, but how the two processes are coordinated is not known. We found that a tissue-wide tension generated by muscle contractions and relayed by tendon-like hemidesmosomes in the dorso-ventral epidermis is required to establish a planar polarity of the apical PAR module in the lateral epidermis. This planar polarized PAR module then controls actin planar organization, thus determining the orientation of cell shape changes and the elongation axis of the whole embryo. This trans-tissular mechanotransduction pathway thus contributes to coordinate the morphogenesis of three embryonic tissues.

developmental biology↗

bioRxiv2017

Tissue-specific activities of the Fat1 cadherin cooperate to control neuromuscular morphogenesis

Muscle morphogenesis is tightly coupled with that of motor neurons (MNs). Both MNs and muscle progenitors simultaneously explore the surrounding tissues while exchanging reciprocal signals to tune their behaviors. We previously identified the Fat1 cadherin as a regulator of muscle morphogenesis, and showed that it is required in the myogenic lineage to control the polarity of progenitor migration. To expand our knowledge on how Fat1 exerts its tissue-morphogenesis regulator activity, we dissected its functions by tissu-specific genetic ablation. An emblematic example of muscle under such morphogenetic control is the cutaneous maximus (CM) muscle, a flat subcutaneous muscle in which progenitor migration is physically separated from the process of myogenic differentiation, but tightly associated with elongating axons of its partner motor neurons. Here, we show that constitutive Fat1 disruption interferes with expansion and differentiation of the CM muscle, with its motor innervation and with specification of its associated MN pool. Fat1 is expressed in muscle progenitors, in associated mesenchymal cells, and in MN subsets including the CM-innervating pool. We identify mesenchyme-derived connective tissue as a cell type in which Fat1 activity is required for the non-cell-autonomous control of CM muscle progenitor spreading, myogenic differentiation, motor innervation, and for motor pool specification. In parallel, Fat1 is required in MNs to promote their axonal growth and specification, indirectly influencing muscle progenitor progression. These results illustrate how Fat1 coordinates the coupling of muscular and neuronal morphogenesis by playing distinct but complementary actions in several cell types.\n\nAuthor summaryFat cadherins are evolutionarily conserved cell adhesion molecules playing key roles in modulating tissue morphogenesis, through the control of collective cell behavior and polarity. We previously identified the mouse Fat1 gene as a regulator of muscle morphogenesis. The present study explores how Fat1 influences neuromuscular morphogenesis in the context of development of a flat subcutaneous muscle, the cutaneous maximus muscle (CM), formed by migratory progenitors emerging from forelimb levels somites, and innervated by a pool of brachial spinal motor neurons (MNs). CM development involves the rostrocaudal planar migration of muscle progenitors and subsequent elongation of muscle fibers to form a fan-shaped muscle. We previously reported that Fat1 was required in muscle progenitors to modulate their migration polarity. Here, these results were expanded by exploring the contribution of Fat1 activities in two other cell types, mesenchymal cells and MNs. We show that Fat1 disruption in connective tissue robustly alters CM muscle morphogenesis, affecting not only progenitor migration and myofiber expansion, but also subsequently impairing axon growth and specification of cognate MNs. In parallel, Fat1 acts in MNs to modulate axonal growth and neuronal specification, modestly influencing muscle morphology. Together, these results show that Fat1 coordinates the coupling between muscle and neuronal development by playing complementary functions in mesenchyme, muscles and MNs. These findings could guide research on muscle pathologies associated with FAT1 alterations in humans.

developmental biology↗

bioRxiv2017

Zebrafish yolk syncytial nuclei migrate along a dynamic microtubule network

In teleosts, the yolk syncytial layer is a multinucleate syncytium that functions as an extraembryonic signaling center to pattern the mesendoderm, coordinate morphogenesis and supply nutrients to the embryo. The zebrafish is an excellent system for studying this morphogenetically active tissue. The external yolk syncytial nuclei (e-YSN) undergo microtubule dependent epiboly movements that distribute the nuclei over the yolk. How e-YSN epiboly proceeds, and what role the yolk microtubule network plays is not understood but currently it is proposed that e-YSN are pulled vegetally as the microtubule network shortens from the vegetal pole. Data from our live imaging studies suggest that the yolk microtubule network is dismantled from the animal and vegetal regions and show that a region of stabilized microtubules forms before nuclear migration begins. e-YSN do not appear to be pulled vegetally but rather move along a dynamic microtubule network. We also show that overexpression of the KASH domain of Syne2a impairs e-YSN movement, implicating the LINC complex in e-YSN migration. This work provides new insights into the role of microtubules in morphogenesis of an extraembryonic tissue.\n\nSummary StatementAnalysis of yolk syncytial nuclear migration during zebrafish epiboly reveals that nuclei migrate along and largely beneath a dynamically yolk microtubule network.

developmental biology↗

bioRxiv2017

Division-independent differentiation mandates proliferative competition among stem cells

Cancer-initiating gatekeeper mutations that arise in stem cells would be especially potent if they stabilize and expand an affected stem lineage (1, 2). It is therefore important to understand how different stem cell organization strategies promote or prevent variant stem cell amplification in response to different types of mutation, including those that activate stem cell proliferation. Stem cell numbers can be maintained constant while producing differentiated products through individually asymmetric division outcomes or by population asymmetry strategies, in which individual stem cell lineages necessarily compete for niche space. We considered alternative mechanisms underlying population asymmetry and used quantitative modeling to predict starkly different consequences of altering proliferation rate: a variant, faster-proliferating mutant stem cell should compete better only when stem cell division and differentiation are independent processes. For most types of stem cell it has not been possible to ascertain experimentally whether division and differentiation are coupled. However, Drosophila Follicle Stem Cells (FSCs) provided a favorable model system to investigate population asymmetry mechanisms and also for measuring the impact of altered proliferation on competition. We found from detailed cell lineage studies that FSC division and FSC differentiation are not coupled. We also found that FSC representation, reflecting maintenance and amplification, was highly responsive to genetic changes that altered only the rate of FSC proliferation. The FSC paradigm therefore provides definitive experimental evidence for the general principle that relative proliferation rate will always be a major determinant of competition among stem cells specifically when stem cell division and differentiation are independent.\n\nSIGNIFICANCEAdult stem cells support tissue maintenance throughout life but they also can be cells of origin for cancer, allowing clonal expansion and long-term maintenance of the first oncogenic mutations. We considered how a mutation that increases the proliferation rate of a stem cell would affect the probability of its competitive survival and amplification for different potential organizations of stem cells. Quantitative modeling showed that the key characteristic predicting the impact of relative proliferation rate on competition is whether differentiation of a stem cell is coupled to its division. We then used Drosophila Follicle Stem Cells to provide definitive experimental evidence for the general prediction that relative proliferation rates dictate stem cell competition specifically for stem cells that exhibit division-independent differentiation.

developmental biology↗

bioRxiv2017

Preovulatory oocyte aging in mice affects fertilization rate and embryonic genome activation

Delayed ovulation, or preovulatory aging, can seriously compromise the developmental competence of oocytes. In the present study, we have investigated the effect of preovulatory aging on preimplantation embryos. Delaying ovulation with the gonadotropin releasing hormone (GnRH) antagonist Cetrorelix led to a decline in 2-cell rate from 76 to 46%. From control mice, an average of 17 embryos per mouse was retrieved. This number decreased to a mean of 5 embryos per mouse after preovulatory aging, suggesting that fertilization is impaired by aging. For analysis of zygotic genome activation, 2-cell embryos were incubated with BrUTP, which was incorporated into nascent RNA and detected by immunohistochemistry. A 2.85-fold increase in fluorescence intensity was detected after aging, pointing to a precocious activation of the genome. A possible effect of preovulatory aging on genomic imprint maintenance was investigated at the 8-cell stage. Deep amplicon bisulfite sequencing of Igf2r, Snrpn, H19 and Pou5f1 showed no significant changes between embryos derived from preovulatory-aged oocytes and control embryos, indicating stable imprint maintenance throughout epigenetic reprogramming. We conclude that preovulatory aging of the oocyte affects fertilization and embryonic genomic activation.

developmental biology↗