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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

Preformed Chromatin Topology Assists TranscriptionalRobustness of Shh during Limb Development

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases proximity coincides with gene activation, whereas in others preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in a loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele resulting in severe Shh loss-of-function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

developmental biology↗

bioRxiv2019

Mechanically activated Piezo channels control outflow tract valve development through Yap1 and Klf2-Notch signaling axis

Mechanical forces are well known for modulating heart valve developmental programs. Yet, it is still unclear how genetic programs and mechanosensation interact during heart valve development. Here, we assessed the mechanosensitive pathways involved during zebrafish outflow tract (OFT) valve development in vivo. Our results show that the hippo effector Yap1, Klf2, and the Notch signaling pathway are all essential for OFT valve morphogenesis in response to mechanical forces, albeit active in different cell layers. Furthermore, we show that Piezo and TRP mechanosensitive channels are essential for regulating these pathways. In addition, live reporters reveal that piezo controls Klf2 and Notch activity in the endothelium and Yap1 expression in the smooth muscle progenitors to coordinate OFT valve morphogenesis. Together, this work identifies a unique morphogenetic program during OFT valve formation and places Piezo as a central modulator of the cell response to forces in this process.

developmental biology↗

bioRxiv2019

PSMC3 is required for spermatogonia niche establishment in mouse spermatogenesis

Males produce millions of spermatozoa each day, which are originated from spermatogonia. Spermatogonia niche establishment and maintenance and the subsequent haploidization of spermatocytes in meiosis are hallmarks of this process. The function of the individual players and coordinated mechanisms regulating different stages of gametogenesis in mammals are not well understood. In this work we focused on the role of PSMC3 in mouse gametogenesis. We observed that Psmc3 is highly expressed in mouse testis, and it is widely expressed in different stages of gamete formation. Conditional deletion of Psmc3 results in both male and female impairment of gonad development at early pre-meiotic stages, but has no apparent effect on meiosis progression. This is likely a consequence of abnormal spermatogonia niche establishment and/or maintenance, revealed by a massive loss of undifferentiated spermatogonia. Our work defines a fundamental role of PSMC3 functions in spermatogenesis during spermatogonia development with direct implications in fertility.

developmental biology↗

bioRxiv2019

Mutations in thyroid hormone receptor α1 cause premature neurogenesis and progenitor cell depletion in human cortical development

Mutations in the thyroid hormone receptor 1 gene (THRA) have recently been identified as a cause of intellectual deficit in humans. Patients present with structural abnormalities including microcephaly, reduced cerebellar volume and decreased axonal density. Here, we show that directed differentiation of THRA mutant patient-derived iPSCs to forebrain neural progenitors is markedly reduced, but mutant progenitor cells can generate deep and upper cortical layer neurons and form functional neuronal networks. Quantitative lineage tracing shows that THRA mutation-containing progenitor cells exit the cell cycle prematurely, resulting in reduced clonal output. Using a novel micropatterned chip assay, we find that spatial self-organisation of mutation-containing progenitor cells is impaired, consistent with downregulated expression of cell-cell adhesion genes. These results reveal for the first time that thyroid hormone receptor 1 is required for normal neural progenitor cell proliferation and organisation in human cerebral cortical development. They also exemplify novel quantitative approaches for studying neurodevelopmental disorders using patient-derived cells in vitro.

developmental biology↗

bioRxiv2019

Structural redundancy in supracellular actomyosin networks enables robust tissue folding

Tissue morphogenesis is strikingly reproducible. Yet, how tissues are robustly sculpted, even under challenging conditions, is unknown. Here, we combined network analysis, experimental perturbations, and computational modeling to determine how network connectivity between hundreds of contractile cells on the ventral side of the Drosophila embryo ensures robust tissue folding. We identified two network properties that mechanically promote robustness. First, redundant supracellular cytoskeletal network paths ensure global connectivity, even with network degradation. By forming many more connections than are required, morphogenesis is not disrupted by local network damage, analogous to the way redundancy guarantees the large-scale function of vasculature and transportation networks. Second, directional stiffening of edges oriented orthogonal to the folding axis promotes furrow formation at lower contractility levels. Structural redundancy and directional network stiffening ensure robust tissue folding with proper orientation.

developmental biology↗

bioRxiv2019

Defining reprogramming checkpoints from single-cell analysis of induced pluripotency

Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a novel combination of epigenetic and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress towards iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play novel functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenetic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

developmental biology↗

bioRxiv2019

Lineage tracing analysis of cone photoreceptor-associated cis-regulatory elements in the developing chicken retina.

During vertebrate retinal development, transient populations of retinal progenitor cells with restricted cell fate choices are formed. One of these progenitor populations expresses the Thrb gene and can be identified with the ThrbCRM1 cis-regulatory element. Short-term assays have concluded that these cells preferentially generate cone photoreceptors and horizontal cells, however developmental timing has precluded an extensive cell type characterization of their progeny. Here we describe the development and validation of a recombinase-based lineage tracing system for the chicken embryo to further characterize the lineage of these cells. The ThrbCRM1 element was found to preferentially form photoreceptors and horizontal cells, as well as a small number of retinal ganglion cells. The photoreceptor cell progeny are exclusively cone photoreceptors and not rod photoreceptors, confirming that ThrbCRM1-progenitor cells are restricted from the rod fate. In addition, specific subtypes of horizontal cells and retinal ganglion cells were overrepresented, suggesting that ThrbCRM1 progenitor cells are not only restricted for cell type, but for cell subtype as well.

developmental biology↗

bioRxiv2019

Yorkie controls tube length and apical barrier integrity in the developing Drosophila airways

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication and apical membrane growth. The Drosophila embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional co-activator of the Hippo pathway, Yorkie (YAP in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water-tightness of tracheal tubes by transcriptional regulation of the enzyme{delta} -aminolevulinate synthase (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality. Short SummaryThis work identified an alternative role of the transcriptional co-activator Yorkie (Yki) in controlling water impermeability and tube size of the developing Drosophila airways. Tracheal impermeability is triggered by Yki-mediated transcriptional regulation of{delta} -aminolevulinate synthase, Alas, whereas tube elongation is controlled by binding of Yki to the actin severing factor Twinstar.

developmental biology↗

bioRxiv2019

Testing models of mRNA localization reveals robustness regulated by reducing transport between cells

Robust control of gene expression in both space and time is of central importance in the regulation of cellular processes, and for multicellular development. However, the mechanisms by which robustness is achieved are generally not identified or well understood. For example, mRNA localization by molecular-motor-driven transport is crucial for cell polarization in numerous contexts, but the regulatory mechanisms that enable this process to take place in the face of noise or significant perturbations are not fully understood. Here we use a combined experimental-theoretical approach to characterize the robustness of gurken/TGF-alpha mRNA localization in Drosophila egg chambers, where the oocyte and 15 surrounding nurse cells are connected in a stereotypic network via intracellular bridges known as ring canals. We construct a mathematical model that encodes simplified descriptions of the range of steps involved in mRNA localization, including production and transport between and within cells until the final destination in the oocyte. Using Bayesian inference, we calibrate this model using quantitative single molecule fluorescence in situ hybridization data. By analyzing both the steady state and dynamic behaviours of the model, we provide estimates for the rates of different steps of the localization process, as well as the extent of directional bias in transport through the ring canals. The model predicts that mRNA synthesis and transport must be tightly balanced to maintain robustness, a prediction which we tested experimentally using an over-expression mutant. Surprisingly, the over-expression mutant fails to display the anticipated degree of overaccumulation of mRNA in the oocyte predicted by the model. Through careful model-based analysis of quantitative data from the over-expression mutant we show evidence of saturation of transport of mRNA through ring canals. We conclude that this saturation engenders robustness of the localization process, in the face of significant variation in the levels of mRNA synthesis. Statement of significanceFor development to function correctly and reliably across a population, gene expression must be controlled robustly in a repeatable manner. How this robustness is achieved is not well understood. We use modelling to better study the localization of polarity determining transcripts (RNA) in fruit fly development. By calibrating our model with quantitative imaging data we are able to make experimentally testable predictions, comparison of which with data from a genetic mutant, reveals evidence that saturation of RNA transport contributes to the robustness of RNA localization.

developmental biology↗

bioRxiv2019

Translational regulation of non-autonomous mitochondrial stress response promotes longevity

Inhibition of mRNA translation delays aging, but the underlying mechanisms remain underexplored. Mutations in both DAF-2 (IGF-1 receptor) and RSKS-1 (ribosomal S6 kinase/S6K) cause synergistic lifespan extension in C. elegans. To understand the roles of S6K-mediated translational regulation in this process, we performed genome-wide translational profiling and genetic screens to identify genes that are not only regulated at the translational level in the daf-2 rsks-1 mutant, but also affect lifespan. Inhibition of CYC-2.1 (cytochrome c) in the germline significantly extends lifespan through non-autonomous activation of the mitochondrial unfolded protein response (UPRmt) and AMP-activated kinase (AMPK) in the metabolic tissue. Furthermore, the RNA-binding protein GLD-1-mediated translational repression of cytochrome c in the germline is important for the non-autonomous activation of UPRmt and synergistic longevity of the daf-2 rsks-1 mutant. Together, these results illustrate a translationally regulated non-autonomous mitochondrial stress response mechanism in the modulation of lifespan by insulin-like signaling and S6K. HighlightsO_LILongevity of the daf-2 rsks-1 mutant is mediated by translational repression of ribosomal proteins and CYC-2.1/cytochrome c. C_LIO_LIGermline inhibition of cyc-2.1 non-autonomously activates UPRmt and AMPK to extend lifespan. C_LIO_LIGLD-1 represses germline cyc-2.1 translation in the daf-2 rsks-1 mutant. C_LIO_LITranslational regulation of cyc-2.1 and UPRmt contribute to the synergistic longevity of the daf-2 rsks-1 mutant. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/533695v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@9f3ce7org.highwire.dtl.DTLVardef@576930org.highwire.dtl.DTLVardef@bb0c09org.highwire.dtl.DTLVardef@133ce4_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

Multiple sources of Shh are critical for the generation and scaling of ventral spinal cord oligodendrocyte precursor populations.

Graded Sonic Hedgehog (Shh) signaling emanating from notochord and floorplate patterns the early neural tube. Soon thereafter, Shh signaling strength within the ventricular zone becomes dis-contiguous and discontinuous along the ventral to dorsal axis suggesting a distribution of Shh that cannot be achieved by diffusion alone. Here we discover that sequential activation of Shh expression by ventricular zone derivatives is critical for counteracting a precocious exhaustion of the Olig2 precursor cell population of the pMN domain at the end of motor neuron genesis and during the subsequent phase of ventral oligodendrocyte precursor production. Selective expression of Shh by motor neurons of the lateral motor column at the beginning of oligodendrogenesis ensures a more yielding pMN domain at limb levels compared to thoracic levels. Thus, patterned expression of Shh by ventricular zone derivatives including earlier born neurons contributes to the scaling of the spinal cord along the anterior - posterior axis by regulating the activity of a select ventricular zone precursor domain at later stages of development.

developmental biology↗

bioRxiv2019

Neuronal differentiation affects tissue mechanics and progenitor arrangement in the vertebrate neuroepithelium

Cell division, movement and differentiation contribute to pattern formation in developing tissues. This is the case in the vertebrate neural tube where neurons differentiate in a characteristic pattern from a highly dynamic proliferating pseudostratified epithelium. To investigate how progenitor proliferation and differentiation affect cell arrangement and growth of the neural tube, we use experimental measurements to develop a mechanical model of the apical surface of the neuroepithelium that incorporates inter-kinetic nuclear movement and spatially varying rates of neuronal differentiation. Simulations predict that tissue growth and the shape of lineage-related clones of cells differ with the rate of differentiation. Growth is isotropic in regions of high differentiation, but dorsoventrally biased in regions of low differentiation. This is consistent with experimental observations. The absence of directional signalling in the simulations indicates that global mechanical constraints are sufficient to explain the observed differences in anisotropy. This provides insight into how the tissue growth rate affects cell dynamics and growth anisotropy and opens up possibilities to study the coupling between mechanics, pattern formation and growth in the neural tube.

developmental biology↗

bioRxiv2019

Multilevel regulation of the glass locus during Drosophila eye development

Development of eye tissue is initiated by a conserved set of transcripton factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control idendity of photoreceptor as well as non-photoreceptors cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintainance of differentiated tissues. The molecular mechanisms that control expression of glass, however remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alterantive small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is surprisingly robust and appears buffered to targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.

developmental biology↗

bioRxiv2019

Fetal stage melanopsin (OPN4) and GNAQ (Gαq) signaling regulates vascular development of the eye

Maturation of sensory systems in mammals is regulated by appropriate sensory stimulation. Developmental refinement of the eye and visual system is regulated by light and visual stimulation. One compelling example is that fetal mouse pups deprived of light exhibit altered vascular development in their eyes. Previous work demonstrated that light activation of the photopigment melanopsin (Opn4), an atypical opsin expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), is crucial to normal vascular development. This suggested the unusual hypothesis that vascular development of the eye was regulated by ipRGC responses in the fetal eye by light that traveled through the body wall of the mother. Here, we test the requirement of OPN4 during fetal stages using genetic approaches. The G-protein GNAQ (Gq) is a candidate mediator of melanopsin signaling. We show that ipRGC-specific deletion of Gnaq phenocopies both hyaloid and retinal vascular development of the Opn4 null mouse. Furthermore, GNAQ gain-of-function in Opn4-expressing cells only during late gestation was sufficient to reverse the consequences for vascular development of either dark rearing or Opn4 loss-of-function. We conclude that melanopsin-dependent signaling in the fetal mouse eye is necessary and sufficient for vascular maturation.

developmental biology↗

bioRxiv2019

Fracking and Ostwald ripening position the lumen of the mouse blastocyst

During mouse preimplantation development, the formation of the blastocoel, a fluid-filled lumen, breaks the radial symmetry of the blastocyst. What controls the formation and positioning of this basolateral lumen remains obscure. We find that accumulation of pressurized fluid fractures cell-cell contacts into hundreds of micron-size lumens. Microlumens eventually discharge their volumes into a single dominant lumen, which we model as a process akin to Ostwald ripening, underlying the coarsening of foams. Using chimeric mutant embryos, we tune the tracking of cell-cell contacts and steer the coarsening of microlumens, allowing us to successfully manipulate the final position of the lumen. We conclude that hydraulic fracture of cell-cell contacts followed by directed coarsening of microlumens sets the first axis of symmetry of the mouse embryo.

developmental biology↗

bioRxiv2019

Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction

Delineating cell lineages is a prerequisite for understanding the genesis of cell types. Recent studies have demonstrated the feasibility of generating and reconstructing CRISPR/Cas9-coded cell lineages. However, these works have not investigated the limitations or optimality of the encoding or reconstruction processes. Here, we surveyed a multitude of reconstruction algorithms and found hierarchical clustering, with a metric based on the number of shared Cas9 edits, provides the best reconstruction. As to the efficiency, the simple encoding method, with constant Cas9/gRNA edit rate, produces exponential reduction in available coding units and severely limits the trackable depth of lineages. To overcome this, we propose alternative encoding methods, one based on parallel gRNA cascades enabled by CLADES, and another based on variable Cas9 editing rate. Both significantly increase the trackable depth. In summary, we provide a theoretical basis in understanding, designing and analyzing efficient and robust CRISPR-based cell lineage tracking system.

developmental biology↗

bioRxiv2019

Identification of genes with enriched expression in early developing mouse cone photoreceptors

Cone photoreceptors are the critical first cells that mediate high acuity vision. Despite their importance and their potential use in cell-based therapies for retinal diseases, there is a lack of knowledge about the early developmental stages of these cells. Here we characterize the expression of the homeobox transcription factor Lhx4 as an early and enriched cone photoreceptor expressed gene in both chicken and mouse. A Lhx4 GFP reporter mouse was found to recapitulate this early cone photoreceptor expression and was used to purify and profile embryonic mouse cone photoreceptors by single cell RNA sequencing. This enrichment in cone photoreceptors allowed for the robust identification of genes associated with the early cone transcriptome and also identified subpopulations of these cells. A comparison to previously reported datasets allowed the classification of genes according to developmental timing, cell type specificity, and whether they were regulated by the rod transcription factor Nrl. This analysis has extended the set of known early cone enriched genes and identified those that are regulated independently of Nrl. This report furthers our knowledge of the transcriptional events that occur in early cone photoreceptors.

developmental biology↗

bioRxiv2019

Single cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progenitors

Development of the vertebrate eye, like many developmental systems, depends on genes that are used iteratively in multiple distinct processes. The OTX2 transcription factor is one such gene, with a requirement for eye formation, photoreceptor formation, and retinal pigment epithelium specification, among others. Recent evidence has suggested that OTX2 is also expressed in subsets of retinal progenitor cells with restricted fate choices. However, given the multiple roles for OTX2 and limitations of conventional conditional knockout strategies, the functional significance of this expression is unknown. Here we use CRISPR/Cas9 gene editing to produce mutations of OTX2, identifying similar phenotypes to those observed in human patients. In addition, we use single cell RNA sequencing to determine the functional consequences of OTX2 gene editing by CRISPR/Cas9 on the population of cells derived from OTX2-expressing retinal progenitor cells. We not only confirm that OTX2 is required for the generation of photoreceptors, but also for maintaining the proliferative potential of cells and suppressing the formation of specific retinal fates. These include subtypes of retinal ganglion and horizontal cells normally associated with these progenitor types, suggesting that in this context OTX2 functions to repress sister cell fate choices. Upregulation of key transcription factors involved in the formation of these cells was observed suggesting that OTX2 is upstream of critical nodes of gene regulatory networks of these alternative fates.

developmental biology↗