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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Multiple functions for the catenin family member plakoglobin in cadherin-dependent adhesion, fibronectin matrix assembly and Xenopus gastrulation movements

Shaping an embryo requires tissue-scale cell rearrangements known as morphogenetic events. These force-dependent processes require cells to adhere to their neighbors, through cadherin-catenin complexes, and to their extracellular matrix substrates, through integrin-based focal contacts. Integrin receptors are not only important for attachment to the extracellular matrix, but also for its fibrillar assembly. Fibrillogenesis requires actomyosin contractility, regulated in part by cadherin-catenin complexes. One such catenin, plakoglobin, mediates the attachment of actin stress fibers to cadherin cytoplasmic tails through its interactions with actin-binding proteins. In Xenopus gastrulae, plakoglobin has been identified as an essential member in the force-induced collective migration of the mesendoderm tissue. In the current study, we have further characterized the role of plakoglobin in two additional morphogenetic processes, epiboly and convergent extension. Plakoglobin-deficient tadpoles are 40% shorter and gastrulae contain notochords that are 60% wider than stage-matched controls, indicating convergent extension defects. The radially intercalating ectoderm of morphant animal caps is nearly twice as thick as controls. Furthermore, morphant embryos exhibit a failure to assemble a fibronectin matrix at the notochord-somite-boundary or along the blastocoel roof. The loss of the fibronectin matrix, while not due to changes in overall patterning, is a result of a failure to assemble the soluble dimers into long fibrils. The force of attachment to a cadherin or fibronectin substrate is reduced in plakoglobin morphants, indicating defects in adhesion to both cadherin and fibronectin. These data suggest that plakoglobin regulates morphogenesis and fibronectin assembly through cell-cell and cell-matrix adhesion.

developmental biology↗

bioRxiv2018

Integrin and ligand-independent PDGFr signaling synergistically contribute to directional migration of Xenopus mesendoderm

Both PDGF signaling and adhesion to fibronectin (FN) matrix have been implicated in the directional collective migration of Xenopus mesendoderm cells at gastrulation. However, mesendoderm explants cultured on FN-coated substrates migrate directionally even in the absence of a source of PDGF. Integrin adhesion has been reported to up-regulate PDGF ligand-independent signaling through the PDGF receptor (PDGFr) in cultured mammalian cells. In order to address whether a similar mechanism stimulates PDGFr signaling in the absence of PDGF-A ligand in amphibian mesendoderm, isolated cells were cultured on bacterial fusion proteins containing the Type-III repeats 9-11 of FN (GST-9.11). Type III9-11 contains the RGD and \"synergy\" (PPSRN) sites required for integrin 5{beta}1 adhesion and activation but lacks the PDGF-A ligand-binding site present in the full-length FN protein. In order to ensure mesendoderm was not exposed to PDGF in vivo prior to removal and culture in vitro, antisense morpholinos were used to inhibit normal expression of PDGF-A ligand in embryos. P-Akt levels were reduced two-fold when either the PDGFr- was knocked down or when cells were plated on GST-9.11a, which contains a point mutation (PPSRN>PPSAN) that prevents both full activation of integrin 5{beta}1 and cell spreading. Reduced expression of PDGFr- was accompanied by perturbations in tissue migration, cytoskeletal organization, polarity of cell protrusions, and focal adhesion area. Mesendoderm cells became rounded, and the actin and cytokeratin filaments appeared collapsed and often colocalized near the cell center. Taken together, these findings suggest that integrin adhesion to FN, acting in synergy with PDGFr-, is sufficient to elevate PI3K-Akt signaling in the mesendoderm even in the absence of the PDGF-A ligand, and to promote forward-directed protrusions and directional tissue migration.

developmental biology↗

bioRxiv2018

Single-cell transcriptome profiling of the Ciona larval brain

The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain.

developmental biology↗

bioRxiv2018

Determination of novel members in the Drosophila melanogaster anterior-posterior patterning system using natural variation

The anterior-posterior axis of the developing Drosophila melanogaster embryo is patterned by a well-studied gene regulatory network called the Gap Gene Network. This network acts to buffer the developing pattern against noise, thereby minimizing errors in gene expression and preventing patterning defects.\n\nIn this paper, we sought to discover novel regulatory regions and transcription factors acting in a subset of the Gap network using a selection of wild-caught fly lines derived from the Drosophila Genetic Reference Panel (DGRP). The fly lines in the DGRP contain subtle genomic differences due to natural variation; we quantified the differences in positioning of gene expression borders of two anterior-poster patterning genes, Kruppel (Kr) and Even-skipped in 13 of the DGRP lines. The differences in the positions of Kruppel and Even-skipped were then correlated to specific single nucleotide polymorphisms and insertions/deletions within the select fly lines. Putative enhancers containing these genomic differences were validated for their ability to produce expression using reporter constructs and analyzed for possible transcription factor binding sites. The identified transcription factors were then perturbed and the resulting Eve and Kr positioning was determined. In this way, we found medea, ultraspiracle, glial cells missing, and orthopedia effect Kr and Eve positioning in subtle ways, while knock-down of pangolin produces significant shifts in Kr and subsequent Eve expression patterns. Most importantly this study points to the existence of many additional novel members that have subtle effects on this system and the degree of complexity that is present in patterning the developing embryo.

developmental biology↗

bioRxiv2018

The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish

The mechanisms that ensure fertilization of eggs by a single sperm are not fully understood. In all teleosts, a channel called the micropyle is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the developing vitelline envelope by a single specialized follicle cell, the micropylar cell, which subsequently degenerates. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish. Genome editing mutants affecting taz can grow to adults, however, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. However, taz mutant eggs have no micropyle. We show that Taz protein is specifically enriched from mid-oogenesis onwards in two follicle cells located at the animal pole of the oocyte, and co-localizes with the actin and tubulin cytoskeleton. Taz protein and micropylar cell are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts.

developmental biology↗

bioRxiv2018

The Germ Theory of Regeneration

Amphibians have remarkable regenerative capabilities, but the mechanisms they use to regenerate are largely unknown. Identifying these mechanism would be of great interest for applications in medicine. Reactive oxygen species (ROS) are continually produced and required during tadpole tail regeneration (1). Production of ROS and tadpole tail regeneration are prevented by NADPH oxidase (Nox) inhibitors, suggesting Nox complexes as the source of ROS. However, the role of ROS and the mechanism of their sustained production throughout regeneration, were not known. NF-{kappa}B is a rapid-acting transcription factor with the potential to dramatically alter the activity and function of a cell (2). NF-{kappa}B is necessary for maintaining the undifferentiated state of human embryonic stem cells (3), human induced pluripotent stem cells (4) and mesenchymal stem cells (5), so may similarly be involved in maintaining the de-differentiated state of regeneration blastema cells. In the absence of an activating signal, NF-{kappa}B is sequestered in the cytoplasm by IkB (inhibitor of NF-{kappa}B), preventing its nuclear localisation and activity. The IkB kinase (IKK) complex inhibits IkB in response to multiple extracellular stimuli, but ROS can also inhibit IkB (6). Nuclear NF-{kappa}B directly activates transcription of several genes encoding Nox proteins (7, 8), so could thereby facilitate ROS production. A positive-feedback loop was hypothesised where ROS inhibit IkB to help maintain continual NF-{kappa}B activity and, in turn, facilitate the continual production of ROS by activating the transcription of Nox-encoding genes. Here we demonstrate the involvement of microorganisms in the initiation of tadpole tail regeneration. Microorganisms offer sources of ligands for toll-like receptor (TLR) pathway activation and consequently, IKK complex activity. It was also suggested that sustained NF-{kappa}B activity allows the continual expression of the genes encoding Nox4 in blastema cells and Nox2 in professional phagocytes. These findings provide potential targets for the activation of regeneration in non-regenerative animals.

developmental biology↗

bioRxiv2018

The potassium channel KCNJ13 is essential for smooth muscle cytoskeletal organization during mouse tracheal tubulogenesis

Tubulogenesis is essential for the formation and function of internal organs. One such organ is the trachea, which allows gas exchange between the external environment and the lungs. However, the cellular and molecular mechanisms underlying tracheal tube development remain poorly understood. Here, we show that the potassium channel KCNJ13 is a critical modulator of tracheal tubulogenesis. We identify Kcnj13 in an ethylnitrosourea forward genetic screen for regulators of mouse respiratory organ development. Kcnj13 mutants exhibit a shorter trachea as well as defective smooth muscle (SM) cell alignment and polarity. KCNJ13 is essential to maintain ion homeostasis in tracheal SM cells, which is required for actin polymerization. This process appears to be mediated, at least in part, through activation of the actin regulator AKT, as pharmacological increase of AKT phosphorylation ameliorates the Kcnj13 mutant trachea phenotypes. These results provide insights into the role of ion homeostasis in cytoskeletal organization during tubulogenesis.

developmental biology↗

bioRxiv2018

Dlk1-Dio3 Locus-Derived LncRNAs Perpetuate Postmitotic Motor Neuron Cell Fate and Subtype Identity

The mammalian imprinted Dlk1-Dio3 locus produces multiple long non-coding RNAs (lncRNAs) from the maternally inherited allele, including Meg3 (i.e. Gtl2) in the mammalian genome. Although this locus has well-characterized functions in stem cell and tumor contexts, its role during neural development is unknown. By transcriptome profiling cell types at each stage of spinal cord development, we uncovered that lncRNAs expressed from the Dlk1-Dio3 locus are predominantly and gradually enriched in rostral motor neurons (MNs). Mechanistically, Meg3 and other Dlk1-Dio3 locus-derived lncRNAs facilitate Jarid2-Ezh2 interactions. Loss of these lncRNAs compromises the H3K27me3 landscape, leading to aberrant expression of progenitor and caudal Hox genes in postmitotic MNs. Our data illustrate that these lncRNAs in the Dlk1-Dio3 locus play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes and they shape MN subtype identity by regulating Hox genes, providing strong evidence of how lncRNAs function during embryonic development.

developmental biology↗

bioRxiv2018

Synchronized mesenchymal cell polarization and differentiation shape the formation of the murine trachea and esophagus

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via elongation and expansion processes. In the first stage, synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.

developmental biology↗

bioRxiv2018

ZAG-1/ZEB and EGL-44/TEAD form a negative feedback loop to safeguard the choice of cell fate

Terminal differentiation generates the specialized features and functions that allow postmitotic cells to acquire their distinguishing characteristics. This process is thought to be controlled by transcription factors called \"terminal selectors\" that directly activate a set of downstream effector genes. In Caenorhabditis elegans the differentiation of both the mechanosensory touch receptor neurons (TRNs) and the multidendritic nociceptor FLP neurons utilize the terminal selectors UNC-86 and MEC-3. The FLP neurons fail to activate TRN genes, however, because a complex of two transcriptional repressors (EGL-44/EGL-46) prevents their expression. Here we show that the ZEB family transcriptional factor ZAG-1 promotes TRN differentiation not by activating TRN genes but by preventing the expression of EGL-44/EGL-46. Since EGL-44/EGL-46 also inhibits the production of ZAG-1, these proteins form a bistable, negative feedback loop that regulates the choice between the two neuronal fates.\n\nSummary statementTranscriptional repressors regulate binary fate choices through reciprocal inhibition during terminal neuronal differentiation. Specifically, ZEB family transcription factor safeguards fate specification of touch receptor neuron by inhibiting TEA domain-containing repressor.

developmental biology↗

bioRxiv2018

Multifunctional RNA-binding proteins mediate ecdysone-dependent germline stem cell self-renewal in Drosophila

Steroid hormones promote stem cell self-renewal in many tissues; however, the molecular mechanisms by which hormone signaling is integrated with niche-derived signals are largely uncharacterized. In the Drosophila ovary, the steroid hormone ecdysone promotes germline stem cell (GSC) self-renewal. Despite strong evidence that ecdysone modulates the reception of bone morphogenetic protein (BMP) signals in GSCs, transcriptional targets of ecdysone signaling that facilitate BMP reception are unknown. Here, we report that ecdysone signaling promotes the expression of the heterogeneous nuclear ribonucleoproteins (hnRNPs) squid, hephaestus, Hrb27C, and Hrb87F in GSCs. These hnRNPs functionally interact with ecdysone signaling to control GSC number and are cell autonomously required in GSCs for their maintenance. We demonstrate that hnRNPs promote GSC self-renewal by binding to transcripts essential for proper BMP signaling, including the BMP receptors thickveins and punt. Our findings support the model that stem cells coordinate local and long-range signals at the transcriptional and post-transcriptional levels to maintain self-renewal in response to physiological demand. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/321109v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1ef6228org.highwire.dtl.DTLVardef@1156460org.highwire.dtl.DTLVardef@170ff15org.highwire.dtl.DTLVardef@17ae36b_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACTEcdysone signaling regulates distinct hnRNPs that bind to BMP signaling targets to control GSC self-renewal. C_FIG SUMMARY STATEMENTEcdysone signaling promotes expression of heterogeneous ribonucleoproteins that modulate BMP-dependent germline stem cell self-renewal in the Drosophila ovary.

developmental biology↗

bioRxiv2018

The osteogenic potential of the neural crest lineage may contribute to craniosynostosis

The craniofacial skeleton is formed from the neural crest and mesodermal lineages, both of which contribute mesenchymal precursors during formation of the skull bones. The large majority of cranial sutures also include a proportion of neural crest derived mesenchyme. While some studies have addressed the relative healing abilities of neural crest and mesodermal bone, relatively little attention has been paid to differences in intrinsic osteogenic potential. Here we use mouse models to compare neural crest osteoblasts (from frontal bones or dura mater) to mesodermal osteoblasts (from parietal bones). Using in vitro culture approaches we find that neural crest-derived osteoblasts readily generate bony nodules while mesodermal osteoblasts do so less efficiently. Furthermore, we find that co-culture of neural crest-derived osteoblasts with mesodermal osteoblasts is sufficient to nucleate ossification centres. All together, this suggests that the intrinsic osteogenic abilities of neural crest-derived mesenchyme may be a primary driver behind craniosynostosis.

developmental biology↗

bioRxiv2018

TET1 drives global DNA demethylation via DPPA3-mediated inhibition of maintenance methylation

Genome-wide DNA demethylation is a unique feature of mammalian development and naive pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naive pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.

developmental biology↗

bioRxiv2018

A re-inducible genetic cascade patterns the anterior-posterior axis of insects in a threshold-free fashion

Gap genes mediate the division of the anterior-posterior axis of insects into different fates through regulating downstream hox genes. Decades of tinkering the segmentation gene network of the long-germ fruit fly Drosophila melanogaster led to the conclusion that gap genes are regulated (at least initially) through a threshold-based French Flag model, guided by both anteriorly- and posteriorly-localized morphogen gradients. In this paper, we show that the expression patterns of gap genes in the intermediate-germ beetle Tribolium castaneum are mediated by a threshold-free Speed Regulation mechanism, in which the speed of a genetic cascade of gap genes is regulated by a posterior gradient of the transcription factor Caudal. We show this by re-inducing the leading gap gene (namely, hunchback) resulting in the re-induction of the gap gene cascade at arbitrary points in time. This demonstrates that the gap gene network is self-regulatory and is primarily under the control of a posterior speed regulator in Tribolium and possibly all insects.

developmental biology↗

bioRxiv2018

Hierarchical organization of developing HSPC in the human embryonic liver

Despite advances to engineer transplantable hematopoietic stem and progenitor cells (HSPCs) for research and therapy, an in depth characterization of the developing human hematopoietic system is still lacking. The human embryonic liver is at the crossroad of several hematopoietic sites and harbours a complex hematopoietic hierarchy including the first, actively dividing, HSPCs that will further seed the definitive hematopoietic organs. However few is known about the hierarchical phenotypic and functional hematopoietic organization operating at these stages of development.\n\nHere, by using a combination of four endothelial and hematopoietic surface markers i.e. the endothelial-specific marker VE-cadherin, the pan-leukocyte antigen CD45, the hemato-endothelial marker CD34 and the Angiotensin-Converting Enzyme (ACE, CD143), encompassing all early human HSPCs, we identified a hematopoietic hierarchy and, among it, a population co-expressing the four markers that uniquely harbored a proliferation and differentiation potential both ex vivo and in vivo. Moreover, we traced back this population to the yolk sac and AGM sites of hematopoietic emergence. Taken together, our data will help to identify human HSPC self-renewal and amplification mechanisms for future cell therapies.\n\nSUMMARY STATEMENTWe uncover the phenotypic and functional hematopoietic hierarchy operating in the early human embryo. It will bring insights into the mechanisms driving hematopoietic stem cell self-renewal for future cell therapies.

developmental biology↗

bioRxiv2018

C. elegans germ cells divide and differentiate along a folded epithelium

Knowing how stem cells and their progeny are positioned within their tissues is essential for understanding their regulation. One paradigm for stem cell regulation is the C. elegans germline, which is maintained by a pool of germline stem cells in the distal gonad, in a region known as the progenitor zone. The C. elegans germline is widely used as a stem cell model, but the cellular architecture of the progenitor zone has been unclear. Here we characterize this architecture by creating virtual 3D models of the progenitor zone in both sexes. We show that the progenitor zone in adult hermaphrodites is essentially a folded epithelium. The progenitor zone in males is not folded. Analysis of germ cell division shows that daughter cells are born side-by-side along the surface of the epithelium. Analysis of a key regulator driving differentiation, GLD-1, shows that germ cells in hermaphrodites differentiate along the path of the folded epithelium, with previously described \"steps\" in GLD-1 expression corresponding to germline folds. Our study provides a three-dimensional view of how C. elegans germ cells progress from stem cell to overt differentiation, with critical implications for regulators driving this transition.

developmental biology↗

bioRxiv2018

Par3 regulates Rac1 signaling and microtubule organization during planar polarization of auditory hair cells

In the inner ear sensory epithelia, hair bundles atop sensory hair cells are mechanosensory apparati with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 plays a key role in planar polarization of hair cells. Par3 deletion in the inner ear resulted in defects in cochlear length, hair bundle orientation and kinocilium positioning. During PCP establishment, Par3 promotes localized Rac-Pak signaling through an interaction with Tiam1. Par3 regulates microtubule dynamics and organization, which is crucial for basal body positioning. Moreover, there is reciprocal regulation of Par3 and the core PCP molecule Vangl2. Thus, we conclude that Par3 is an effector and integrator of cell-intrinsic and tissue-level PCP signaling.\n\nOne sentence summaryPar3 regulates planar polarity of auditory hair cells

developmental biology↗

bioRxiv2018

Simultaneous in vivo time-lapse stiffness mapping and fluorescence imaging of developing tissue

Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that in the developing Xenopus brain, a stiffness gradient evolves over time because of differential cell proliferation. Subsequently, axons turn to follow this gradient, underpinning the importance of time-resolved mechanics measurements.

developmental biology↗