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Results for “Developmental Biology”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

GSK3 Controls Migration of the Neural Crest Lineage

Migration of the neural crest lineage is critical to its physiological function. Mechanisms controlling neural crest migration are comparatively unknown, due to difficulties accessing this cell population in vivo. Here, we uncover novel requirements of glycogen synthase kinase 3 (GSK3) in regulating the neural crest. We demonstrate that GSK3 is tyrosine phosphorylated (pY) in neural crest cells and that this activation depends on anaplastic lymphoma kinase (ALK), a protein associated with neuroblastoma. Consistent with this, neuroblastoma cells with pathologically increased ALK activity express high levels of pY-GSK3 and migration of these cells can be inhibited by GSK3 or ALK blockade. In normal neural crest cells, loss of GSK3 leads to increased pFAK and misregulation of Rac1 and lamellipodin, key regulators of cell migration. Genetic reduction of GSK-3 results in failure of migration. All together, this work identifies a role for GSK3 in cell migration during neural crest development and cancer.

developmental biology↗

bioRxiv2018

The chick caudo-lateral epiblast acts as a permissive niche for generating neuromesodermal progenitor behaviours

Neuromesodermal progenitors (NMps) are a population of bipotent progenitors that maintain competence to generate both spinal cord and paraxial mesoderm throughout the elongation of the posterior body axis. Recent studies have generated populations of NMp-like cells in culture and have been shown to differentiate to both neural and mesodermal cell fates when transplanted into either mouse or chick embryos. Here, we aim to compare the potential of mouse embryonic stem (ES) cell-derived progenitor populations to generate NMp behavior against both undifferentiated and differentiated populations. We define NMp behaviour as the ability of cells to i) contribute to a significant proportion of the anterior-posterior body axis, ii) enter into both posterior neural and somitic compartments and, iii) retain a proportion of the progenitor population within the posterior growth zone. We compare previously identified ES cell-derived NMp-like populations to undifferentiated mouse ES cells and find that they all display similar potentials to generate NMp behaviour in vivo. To assess whether this competence is lost upon further differentiation, we generated anterior and posterior embryonic cell types through the generation of 3D gastruloids and show that NMp competence is lost within the anterior (Brachyury negative) portion of the gastruloid. Taken together, this demonstrates that the chick caudo-lateral epiblast is a highly permissive environment for testing NMp competence and is therefore not suitable as a positive test of neuromesodermal progenitor identity (ie. specification). However, it does act as an appropriate system to test for the loss of NMp potential, and therefore offers insight as a functional test for the regulation of NMp competence in vivo.

developmental biology↗

bioRxiv2018

Wolbachia control stem cell behavior and stimulate germline proliferation in filarial nematodes

Although symbiotic interactions are ubiquitous in the living world, examples of developmental symbioses are still scarce. We show here the crucial role of Wolbachia in the oogenesis of filarial nematodes, a class of parasites of biomedical and veterinary relevance. While the Wolbachia-depleted nematodes produce faulty embryos, we identified thanks to newly generated techniques the earliest requirements of Wolbachia in the germline. They stimulate its proliferation in a cell-autonomous manner, in parallel of the known key controllers, and not through nucleotide supplementation as previously hypothesized. We also found Wolbachia to maintain the quiescence of a pool of germline stem cells ensuring for many years a constant delivery of about 1400 eggs per day. The loss of quiescence upon Wolbachia depletion, as well as the disorganization of the distal germline suggest that Wolbachia are required to execute the proper germline stem cell developmental program in order to produce viable eggs and embryos.

developmental biology↗

bioRxiv2018

Insect wings and body wall evolved from ancient leg segments

The origin of insect wings has long been debated. Central to this debate is whether wings evolved from an epipod (outgrowth, e.g., a gill) on ancestral crustacean leg segments, or represent a novel outgrowth from the dorsal body wall that co-opted some of the genes used to pattern the epipods. To determine whether wings can be traced to ancestral, pre-insect structures, or arose by co-option, comparisons are necessary between insects and arthropods more representative of the ancestral state, where the hypothesized proximal leg region is not fused to the body wall. To do so, we examined the function of five leg patterning genes in the crustacean Parhyale hawaiensis and compared this to previous functional data from insects. By comparing gene knockout phenotypes of leg patterning genes in a crustacean with those of insects, we show that two ancestral crustacean leg segments were incorporated into the insect body, moving the legs epipod dorsally, up onto the back to form insect wings. Thus, our data shows that much of the body wall of insects, including the entire wing, is derived from these two ancestral proximal leg segments. This model explains all observations in favor of either the body wall origin or proximal leg origin of insect wings. Thus, our results show that insect wings are not novel structures, but instead evolved from existing, ancestral structures.\n\nOne Sentence SummaryCRISPR-Cas9 knockout of leg gap genes in a crustacean reveals that insect wings are not novel structures, they evolved from crustacean leg segments

developmental biology↗

bioRxiv2018

A three-dimensional staging system of mouse endometrial gland morphogenesis

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, development, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus that occurs after birth. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Subsequently, the developing uterine glands invade into the adjacent stroma. Mouse uterine gland morphology is poorly understood because it is based on two-dimensional (2D) histological observations. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior-posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during postnatal development in the mouse. We define 6 stages: Stage 0: Aglandular, Stage 1: Bud, Stage 2: Teardrop, Stage 3: Elongated, Stage 4: Sinuous, and Stage 5: Primary Branches. This staging system provides a standardized key to assess and quantify uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous.

developmental biology↗

bioRxiv2018

Multiple sources of variability drive a stochastic cell fate decision in C. elegans

During development, cell fate decisions are often highly stochastic, but with the frequency of the different possible fates tightly controlled. To understand how signaling networks control the cell fate frequency of such random decisions, we studied the stochastic decision of the Caenorhabditis elegans P3.p cell to either fuse to the hypodermis or assume vulva precursor cell fate. Using time-lapse microscopy to measure the single-cell dynamics of two key inhibitors of cell fusion, the Hox gene LIN-39 and Wnt signaling through the {beta}-catenin BAR-1, we uncovered significant variability in the dynamics of LIN-39 and BAR-1 levels. Most strikingly, we observed that BAR-1 accumulated in a single, 1-4 hour pulse at the time of the P3.p cell fate decision, with strong variability both in pulse slope and time of pulse onset. We found that the time of BAR-1 pulse onset was delayed relative to the time of cell fusion in mutants with low cell fusion frequency, linking BAR-1 pulse timing to cell fate outcome. Overall, a model emerged where animal-to-animal variability in LIN-39 levels and BAR-1 pulse dynamics biases cell fate by modulating their absolute level at the time cell fusion is induced. Our results highlight that timing of cell signaling dynamics, rather than its average level or amplitude, could play an instructive role in determining cell fate.\n\nArticle summaryWe studied the stochastic decision of the Caenorhabditis elegans P3.p cell to either fuse to the hypodermis or assume vulva precursor cell fate. We uncovered significant variability in the dynamics of LIN-39/Hox and BAR-1/{beta}-catenin levels, two key inhibitors of cell fusion. Surprisingly, we observed that BAR-1 accumulated in a 1-4 hour pulse at the time of the P3.p cell fate decision, with variable pulse slope and time of pulse onset. Our work suggests a model where animal-to-animal variability in LIN-39 levels and BAR-1 pulse dynamics biases cell fate by modulating their absolute level at the time cell fusion is induced.

developmental biology↗

bioRxiv2018

CDK1 and CDK2 regulate phosphorylation-dependent NICD1 turnover and the periodicity of the segmentation clock

All vertebrates share a segmented body axis. Segments form periodically from the rostral end of the presomitic mesoderm (PSM) and this periodicity is regulated by the segmentation clock, a molecular oscillator that drives dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. However, the mechanistic details of how NICD stability is regulated are unclear.\n\nWe identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle-dependent manner. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations.

developmental biology↗

bioRxiv2018

Quantification of gene expression patterns to reveal the origins of abnormal morphogenesis

The earliest developmental origins of dysmorphologies are poorly understood in many congenital diseases. They often remain elusive because the first signs of genetic misregulation may initiate as subtle changes in gene expression, which can be obscured later in development due to secondary phenotypic effects. We here develop a method to trace back the origins of phenotypic abnormalities by accurately quantifying the 3D spatial distribution of gene expression domains in developing organs. By applying geometric morphometrics to 3D gene expression data obtained by Optical Projection Tomography, our approach is sensitive enough to find regulatory abnormalities never previously detected. We identified subtle but significant differences in gene expression of a downstream target of the Fgfr2 mutation associated with Apert syndrome. Challenging previous reports, we demonstrate that Apert syndrome mouse models can further our understanding of limb defects in the human condition. Our method can be applied to other organ systems and models to investigate the etiology of malformations.

developmental biology↗

bioRxiv2018

Efficient production of erythroid, megakaryoid and myeloid cells, using a single cell-derived iPSC colony differentiation

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryoid and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications.\n\nHighlightsO_LIEfficient hematopoietic differentiation from single cell-derived iPSC colonies\nC_LIO_LIReproducible feeder-free, monolayer differentiation system independent of iPSC line\nC_LIO_LIProduction of erythroid, megakaryoid and myeloid cells with high-purity\nC_LIO_LIPlatform for hematopoietic developmental research and future clinical application\nC_LI

developmental biology↗

bioRxiv2018

Neonate gut and respiratory microbiota: coordinated development through time and space

Background: Postnatal development of the microbiota in early life influences immunity, metabolism, neurodevelopment and long-term infant health. Microbiome development occurs at multiple body sites, each with distinct community compositions and functions. Associations between microbiota at multiple sites represent an unexplored influence on the infant microbiome. Here, we examined co-occurrence patterns of gut and respiratory microbiota in pre- and full-term infants over the first year of life, a period critical to neonatal development and risk of respiratory diseases.\n\nResults: Gut and respiratory microbiota collected as longitudinal rectal, throat and nasal samples from 38 pre-term and 44 full-term infants were first clustered into community state types (CSTs) on the basis of their composition. Multiple methods were used to relate the occurrence of CSTs to several measures of infant maturity, including gestational age (GA) at birth, week of life (WOL), and post menstrual age (PMA: equal to GA plus WOL). Manifestation of CSTs followed one of three patterns with respect to infant maturity. First, chronological: independent of infant maturity (GA) at birth, and strongly associated with post-natal age (WOL). Second, idiosyncratic: primarily dependent on maturity (GA) at birth, with persistent differences in CST occurrence between pre- and full-term infants through the first year of life. Third, convergent: CSTs appear earlier in infants with greater maturity (GA) at birth, but after a sufficient post-natal interval their occurrence in pre-term infants reaches parity with full-term infants. The composition of CSTs was highly dissimilar between different body sites, but the CST of any one body site was highly predictive of the CSTs at other body sites. There were significant associations between the abundance of individual taxa at each body site and the CSTs of the other body sites, which persisted after stringent control for the non-linear effects of infant maturity. Significant canonical correlations exist between the microbiota composition at each pair of body sites, with the strongest correlations between more proximal locations.\n\nConclusion: Cross-body site associations of developing infant microbiota suggest the importance of research and clinical practices that focus on dynamic interactions between multiple microbial communities to elucidate and promote systemic microbiota development.

developmental biology↗

bioRxiv2018

Zebrabase: An intuitive tracking solution for aquatic model organisms

Small fish species, like zebrafish or medaka, are constantly gaining popularity in basic research and disease modeling as a useful alternative to rodent model organisms. However, the tracking options for fish within a facility are rather limited. Here, we present an aquatic species tracking database, Zebrabase, developed in our zebrafish research and breeding facility that represents a practical and scalable solution and an intuitive platform for scientists, fish managers and caretakers, in both small and large facilities. Zebrabase is a scalable, crossplatform fish tracking database developed especially for research fish facilities but can be easily adapted for a wide variety of aquatic model organisms housed in tanks. It provides sophisticated tracking, reporting, and management functions that help keep the animalrelated records well-organized including a QR code functionality for tank labeling. The implementation of various user roles ensures a functional hierarchy and customized access to specific functions and data. In addition, Zebrabase enables easy personalization of rooms and racks, and its advanced statistics and reporting options make it an excellent tool for creating periodic reports of animal usage and productivity. The communication between the facility and researchers can be streamlined by the requesting capabilities of the database. Finally, Zebrabase also features an interactive breeding history and a smart interface with advanced visualizations and intuitive color coding allowing to speed up the processes.

developmental biology↗

bioRxiv2018

Prenatal aromatase inhibition alters postnatal immune function in domestic chickens (Gallus gallus)

In birds, exposure to testosterone during embryonic development can suppress immune function; however, it is unclear whether this is caused by direct stimulation of androgen receptors. Estradiol is synthesized from testosterone by the enzyme aromatase, and this conversion is a necessary step in many signaling pathways that are ostensibly testosterone-dependent. Many lines of evidence in mammals indicate that estradiol can affect immune function. We tested the hypothesis that immunosuppressive effects of avian in ovo testosterone exposure are mediated by conversion to estradiol by aromatase, using Fadrozole to inhibit aromatization of endogenous testosterone during a crucial period of embryonic immune system development in domestic chickens (Gallus gallus). We then measured total IgY antibody count, response to PHA challenge, mass of thymus and bursa of Fabricius, and plasma testosterone post-hatch on days 3 and 18. We predicted that if immunomodulation by testosterone is dependent on aromatization, then Fadrozole treatment would lead to elevated immune activity by inhibiting estrogen production. Conversely, if testosterone inhibits immune function directly by binding to androgen receptors, then Fadrozole treatment would likely not alter immune function. Fadrozole treated birds had decreased day 3 plasma IgY antibody titers but there was a strong trend towards increased day 18 thymic mass. Furthermore, Fadrozole treatment generated a positive relationship between testosterone and thymic mass in males, and tended to increase day 18 IgY levels for a given bursal mass in females. There was no effect on PHA response, bursal mass, or plasma testosterone at either age. Overall, Fadrozole treated birds tended to have elevated indicators of immune function, implicating aromatization as a relevant pathway through which developmental exposure to testosterone can affect immunity.

developmental biology↗

bioRxiv2018

PRC1-dependent compaction of Hox gene clusters prevents transcriptional derepression during early Drosophila embryogenesis

Summary paragraphPolycomb-group (PcG) proteins are conserved chromatin factors that maintain the silencing of key developmental genes, notably the Hox gene clusters, outside of their expression domains [1-3]. Polycomb repressive complex 2 (PRC2) trimethylates lysine K27 of histone H3 [4], and PRC1 collaborates with PRC2 in gene silencing. Genome-wide studies have revealed large H3K27me3 chromatin domains bound by PcG proteins, and Polycomb domains fold into distinct nuclear structures [5-9]. Although PRC1 is involved in chromatin compaction [10-16], it is unknown whether PRC1-dependent transcriptional silencing is a consequence of its role on higher-order chromatin folding. This is because depletion of PRC1 proteins typically induces both chromatin unfolding and ectopic transcription, and ectopic transcription can open chromatin by itself. To disentangle these two components, we analysed the temporal effects of two PRC1 proteins, Polyhomeotic (Ph) and Polycomb (Pc), on Hox gene clusters during Drosophila embryogenesis. We show that the absence of Ph or Pc affects the higher-order chromatin folding of Hox clusters prior to ectopic Hox gene transcription, demonstrating that PRC1 primary function during early embryogenesis is to compact its target chromatin. During later embryogenesis, we observed further chromatin opening at Hox complexes in both Ph and Pc mutants, which was coupled to strong deregulation of Hox genes at this stage of development. Moreover, the differential effects of Ph and Pc on Hox cluster folding matches the differences in ectopic Hox gene expression observed in these two mutants, suggesting that the degree of Hox derepression in PcG mutants depends on the degree of structural constraints imposed by each PcG component. In summary, our data demonstrate that binding of PRC1 to large genomic domains during early embryogenesis induces the formation of compact chromatin to prevent ectopic gene expression at later time-points. Thus, epigenetic mechanisms such as Polycomb mediated silencing act by folding chromatin domains and impose an architectural layer to gene regulation.

developmental biology↗

bioRxiv2018

RNA polymerase II pausing regulates a quiescence-dependent transcriptional program, priming cells for cell cycle reentry

Adult stem cells persist in mammalian tissues by entering a state of reversible arrest or quiescence associated with low transcription. Using cultured myoblasts and primary muscle stem cells, we show that RNA synthesis is strongly repressed in G0, returning within minutes of activation. We investigate the underlying mechanism and reveal a role for promoter-proximal RNAPol II pausing: by mapping global Pol II occupancy using ChIP-seq, in conjunction with RNA-seq to identify repressed transcriptional networks unique to G0. Strikingly, Pol II pausing is enhanced in G0 on genes encoding regulators of RNA biogenesis (Ncl, Rps24, Ctdp1), and release of pausing is critical for cell cycle re-entry. Finally, we uncover a novel, unexpected repressive role of the super-elongation complex component Aff4 in G0-specific stalling. We propose a model wherein Pol II pausing restrains transcription to maintain G0, preconfigures gene networks required for the G0-G1 transition, and sets the timing of their transcriptional activation.

developmental biology↗

bioRxiv2018

Basal constriction during midbrain-hindbrain boundary morphogenesis is mediated by Wnt5b and Focal Adhesion Kinase.

Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3{beta} overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak is epistatic to wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.

developmental biology↗

bioRxiv2018

Bovine mammary gland development: new insights into the epithelial hierarchy

Milk production is highly dependent on the extensive development of the mammary epithelium, which occurs during puberty. It is therefore essential to distinguish the epithelial cells committed to development during this key stage from the related epithelial hierarchy. Using cell phenotyping and sorting, we highlighted three sub-populations that we assume to be progenitors. The CD49fhighCD24neg cells expressing KRT14, vimentin and PROCR corresponded to basal progenitors whereas the CD49flowCD24neg cells expressing luminal KRT, progesterone and prolactin receptors, were of luminal lineage. The CD49flowCD24pos cells had features of a dual lineage, with luminal and basal characteristics (CD10, ALDH1 and KRT7 expression) and were considered to be early common (bipotent) progenitors. The mammary stem cell (MaSC) fraction was recovered in a fourth sub-population of CD49fhighCD24pos cells that expressed CD10/KRT14 and KRT7. The differential ALDH1 activities observed within the MaSC fraction allowed to discriminate between two states: quiescent MaSCs and lineage-restricted \"activated\" MaSCs. The in-depth characterization of these epithelial sub-populations provides new insights into the epithelial cell hierarchy in the bovine mammary gland and suggests a common developmental hierarchy in mammals.

developmental biology↗

bioRxiv2018

Feedback control of neurogenesis by tissue packing

Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Whilst many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find that dense tissue packing and complex cell geometries play a significant role in regulating differentiation rate in the zebrafish neural tube. Specifically, in regions of high cell density, progenitors are physically pushed away from the apical surface, which, in a Notch-dependent manner, leads to their differentiation. Using simulations we show that this naturally performs negative feedback control on cell number. Our results suggest a model whereby differentiation rate is carefully tuned to correct fluctuations in cell number, originating from variable cell cycle progression and inherently probabilistic differentiation programs.

developmental biology↗

bioRxiv2018

Chiral cilia orientation in the left-right organizer

Chirality is a property of asymmetry between an object and its mirror image. Most biomolecules and cells are intrinsically chiral. Whether cellular chirality can be transferred to asymmetry at the tissue scale remains an unresolved issue. This question is particularly relevant in the left-right organizer (LRO), where cilia motility and chiral flow are thought to be the main drivers of left-right axis symmetry breaking. Here, we built a quantitative approach based on live imaging to set apart the contributions of various pathways to the spatial orientation of cilia in the Kupffers vesicle (KV, zebrafish LRO). We found that cilia populating the zebrafish LRO display an asymmetric orientation between the right and left side of the LRO. Cilia orientations, therefore, give the KV cells a sense of chirality which is different from the chirality of cilia rotation. Surprisingly, we found this asymmetry does not depend on the left-right signalling pathway or flow. Furthermore, we show the establishment of the chirality is dynamic and depends on planar cell polarity. Together, this work identifies a different type of asymmetry in the LRO and sheds light on the complexity of chirality genesis in developing tissues.

developmental biology↗