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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Comprehensive analysis of retinal development at single cell resolution identifies NFI factors as essential for mitotic exit and specification of late-born cells.

Precise temporal control of gene expression in neuronal progenitors is necessary for correct regulation of neurogenesis and cell fate specification. However, the extensive cellular heterogeneity of the developing CNS has posed a major obstacle to identifying the gene regulatory networks that control these processes. To address this, we used single cell RNA-sequencing to profile ten developmental stages encompassing the full course of retinal neurogenesis. This allowed us to comprehensively characterize changes in gene expression that occur during initiation of neurogenesis, changes in developmental competence, and specification and differentiation of each of the major retinal cell types. These data identify transitions in gene expression between early and late-stage retinal progenitors, as well as a classification of neurogenic progenitors. We identify here the NFI family of transcription factors (Nfia, Nfib, and Nfix) as genes with enriched expression within late RPCs, and show they are regulators of bipolar interneuron and Muller glia specification and the control of proliferative quiescence.

developmental biology↗

bioRxiv2018

A test of the general occurrence and predictive utility of isochronal, equiproportional, ‘variable proportional’, and ‘mixed’ development among arthropods

In isochronal (ICD) and equiproportional development (EPD), the proportion of total immature (egg, larval, and/or juvenile) development spent in each stage (developmental proportion) does not vary among stages or temperatures, respectively. ICD and EPD have mainly been reported in copepods, and whether they occur in other arthropods is not known. If they did, then rearing studies could be simplified because the durations of later developmental stages could be predicted based on those of earlier ones. The goal of this study was to test whether different taxa have ICD, EPD, or an alternative development type in which stage-specific proportions depend on temperature, termed variable proportional development (VPD), and also how well each development type allowed later-stage durations to be predicted from earlier ones. Data for 71 arthropods (arachnids, copepod and decapod crustaceans, and insects) were tested, and most (85.9 %) species were concluded to have VPD, meaning that ICD and EPD do not occur generally. However, EPD predicted later-stage durations comparably well to VPD (within 19-23 %), and thus may still be useful. Interestingly, some species showed a mixed form of development, where some stages developmental proportions varied with temperature while those of others did not, which should be further investigated.\n\nHighlightsO_LIWhether arthropod development is generally isochronal or equiproportional was tested\nC_LIO_LIDevelopmental proportions of most species stages varied with temperature\nC_LIO_LIMany species had mixed development between variable and equiproportional types\nC_LIO_LIThe general occurrence of isochronal and equiproportional development was rejected\nC_LIO_LIEquiproportional development did make reasonable predictions of stage durations\nC_LI

developmental biology↗

bioRxiv2018

Evolution of limb development in cephalopod mollusks

Cephalopod mollusks evolved numerous anatomical innovations, including specialized arms and tentacles, but little is known about the developmental mechanisms underlying the evolution of cephalopod limbs. Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods, although they evolved limbs independently. In cuttlefish limb buds, Hedgehog is expressed anteriorly. Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications. Bmp and Wnt signaling, which establishes dorsoventral polarity in vertebrate and arthropod limbs, is similarly polarized in cuttlefish. Inhibition of the dorsal Bmp signal caused ectopic expression of Notum, a ventral sucker field marker, and development of ectopic suckers. Cuttlefish limbs also show proximodistally regionalized expression of Htx, Exd, Dll, Dac, Sp8/9, and Wnt genes, which delineate arm and tentacle sucker fields. These results suggest that cephalopod limbs evolved by parallel activation of an ancient genetic program for appendage development that was present in the bilaterian common ancestor.

developmental biology↗

bioRxiv2018

Mutation of sequences flanking and separating transcription factor binding sites in a Drosophila enhancer significantly alter its output

Here we explore how mutating different sequences in an enhancer that regulates patterned gene expression in Drosophila melanogaster embryos can affect its output. We used quantitative imaging to analyze the effects of a wide variety of mutations in the hunchback distal anterior enhancer. This enhancer has been shown to respond to the anterior morphogen Bicoid, but we found that mutations in only one of the five strong Bicoid sites in the enhancer has a significant effect on its binding. The pioneer factor Zelda, which binds to this enhancer and is the only other factor implicated in its activity besides Bicoid. However, we found that mutations of all its sites only has modest effect that is limited to reduction of its output in more posterior regions of the embryo, where Bicoid levels are low. In contrast to the modest effects of mutating known transcription factor binding sites, randomizing the sequences between Zelda and Bicoid sites significantly compromised enhancer activity. Finer mapping suggested that the sequences that determine activity are broadly distributed in the enhancer. Mutations in short sequences flanking Bicoid binding sites have stronger effects than mutations to Bicoid sites themselves, highlighting the complex and counterintuitive nature of the relationship between enhancer sequence and activity.

developmental biology↗

bioRxiv2018

Activin signaling informs the graded pattern of terminal mitosis and hair cell differentiation in the mammalian cochlea

The mammalian auditory sensory epithelium has one of the most stereotyped cellular patterns known in vertebrates. Mechano-sensory hair cells are arranged in precise rows, with one row of inner and three rows of outer hair cells spanning the length of the spiral-shaped sensory epithelium. Aiding such precise cellular patterning, differentiation of the auditory sensory epithelium is precisely timed and follows a steep longitudinal gradient. The molecular signals that promote auditory sensory differentiation and instruct its graded pattern are largely unknown. Here, we identify Activin A as an activator of hair cell differentiation and show, using mouse genetic approaches, that a local gradient of Activin A signaling within the auditory sensory epithelium times the longitudinal gradient of hair cell differentiation. Furthermore, we provide evidence that Activin-type signaling regulates a radial gradient of terminal mitosis within the auditory sensory epithelium, which constitutes a novel mechanism for limiting the number of inner hair cells being produced.

developmental biology↗

bioRxiv2018

Damage-induced reactive oxygen species enable regenerative signalling by the rapid repositioning of Hedgehog expressing cells.

Aquatic vertebrates have a remarkable ability to regenerate limbs and tails after amputation. Previous studies indicate that reactive oxygen species (ROS) signaling initiates regeneration, but the mechanism by which this takes place is poorly understood. Developmental signalling pathways have been shown to have pro-regenerative roles in many systems. However, whether these are playing roles that are specific to regeneration, or are simply recapitulating their developmental functions is unclear. We have analysed zebrafish larval tail regeneration and find evidence that ROS released upon wounding cause repositioning of notochord cells to the damage site. These cells secrete Hedgehog ligands which are required for regeneration. Hedgehog signalling is not required for normal tail development suggesting that it has a regeneration specific role. Our results provide a model for how ROS initiate tail regeneration, and indicate that developmental signaling pathways can play regenerative functions that are not directly related to their developmental roles.

developmental biology↗

bioRxiv2018

Choline Transporter in α/β core neurons of Drosophila mushroom body non-canonically regulates pupal eclosion and maintains neuromuscular junction integrity

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with the different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called /{beta} core (/{beta}c) neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in /{beta}c neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.

developmental biology↗

bioRxiv2018

Unique morphogenetic signatures define mammalian neck muscles and associated connective tissues

In vertebrates, head and trunk muscles develop from different mesodermal populations and are regulated by distinct genetic networks. Neck muscles at the head-trunk interface remain poorly defined due to their complex morphogenesis and dual mesodermal origins. Here, we use genetically modified mice to establish a 3D model that integrates regulatory genes, cell populations and morphogenetic events that define this transition zone. We show that the evolutionary conserved cucullaris-derived muscles originate from posterior cardiopharyngeal mesoderm, not lateral plate mesoderm, and we define new boundaries for neural crest and mesodermal contributions to neck connective tissue. Furthermore, lineage studies and functional analysis of Tbx1- and Pax3-null mice reveal a unique genetic program for somitic neck muscles that is distinct from that of somitic trunk muscles. Our findings unveil the embryological and developmental requirements underlying tetrapod neck myogenesis and provide a blueprint to investigate how muscle subsets are selectively affected in some human myopathies.

developmental biology↗

bioRxiv2018

Zelda potentiates transcription factor binding to zygotic enhancers by increasing local chromatin accessibility during early Drosophila melanogaster embryogenesis.

The maternally deposited transcription factor Zelda binds to and is required for the activation of a large number of genes in early Drosophila development, and has been suggested to act as a pioneer factor. In this study, we investigated the temporal dynamics of Zelda binding along with the maternal patterning factors Dorsal and Caudal during early embryogenesis. We found in regions bound by Zelda and either Dorsal or Caudal, Zelda binding was detected, and reached maximum levels, earlier than Caudal and Dorsal, providing support of its role as a pioneer factor. We found that Dorsal and Caudal binding correlated strongly with Zelda binding at mitotic cycle 12, suggesting that Zelda is important for early binding by these factors and early onset of their target gene expression. At the same time, we show that among Dorsal target enhancers, the dorsal and ventral ectoderm enhancers are much more strongly associated with Zelda than mesoderm enhancers, revealing an additional function of Zelda in coordinating spatial activity of enhancers. We have also investigated the role of Zelda on chromatin structure. We found that in early embryos, before Dorsal and Caudal are bound at significant levels, Zelda binding is associated with histone acetylation and local histone depletion. These chromatin associated changes accompanied with increased local chromatin accessibility were also detected around Zelda peaks in coding sequences that do not appear to play a role in subsequent transcription factor binding. These findings suggest that Zelda binding itself can lead to chromatin structural changes. Finally, we found that Zelda motifs, both bound and unbound, tend to be associated with positioned nucleosomes, which we suggest may be important for the regulatory specificity of enhancers.

developmental biology↗

bioRxiv2018

Ral signals through a MAP4 Kinase-p38 MAP kinase cascade in C. elegans cell fate patterning.

C. elegans vulval precursor cell (VPC) fates are patterned by an EGF gradient. High dose EGF induces 1{degrees} VPC fate, while lower dose EGF contributes to 2{degrees} fate in support of LIN-12/Notch. We previously showed that the EGF 2{degrees}-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK MAP kinase cascade that promotes 1{degrees} fate to the non-canonical RalGEF-Ral that promotes 2{degrees} fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well-understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a CNH domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2{degrees} fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.

developmental biology↗

bioRxiv2018

Single-cell RNA-seq reveals distinct dynamic behavior of sex chromosomes during early human embryogenesis

AbstractO_ST_ABSBackgroundC_ST_ABSSeveral animal and human studies have demonstrated that sex affects kinetics and metabolism during early embryo development. However, the mechanism governing these differences at the molecular level is unknown, warranting a systematic profiling of gene expression in males and females during embryogenesis.\n\nFindingsWe performed comprehensive analyses of gene expression comparing male and female embryos using available single-cell RNA-sequencing data of 1607 individual cells from 99 human preimplantation embryos, covering development stages from 4-cell to late blastocyst (E2 to E7). Consistent chromosome-wide transcription of autosomes was observed, while sex chromosomes showed significant differences after embryonic genome activation (EGA). Differentially expressed genes (DE genes) in male and female embryos mainly involved in the cell cycle, protein translation and metabolism. The Y chromosome was initially activated by pioneer genes, RPS4Y1 and DDX3Y, while the two X chromosomes in female were widely activated after EGA. Expression of X-linked genes in female significantly declined at the late blastocyst stage, especially in trophectoderm cells, revealing a rapid process of dosage compensation.\n\nConclusionsWe observed imbalanced expression from sex chromosomes in male and female embryos during EGA, with dosage compensation occurring first in female trophectoderm cells. Studying the effect of sex differences during human embryogenesis, as well as understanding the mechanism of X chromosome inactivation and its correlation with early miscarriage, will provide a basis for advancing assisted reproductive technology (ART) and thereby improve the treatment of infertility and possibly enhance reproductive health.

developmental biology↗

bioRxiv2018

Nematode germ granule assembly is linked to mRNA repression

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P-granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P-granule scaffolding protein, called PGL, to investigate the functional relationship between P-granule assembly and function. Using a protein-RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL granules. We determine the crystal structure of the PGL N-terminal region to 1.5 [A], discover its dimerization and identify key residues at the dimer interface. In vivo mutations of those interface residues prevent P-granule assembly, de-repress PGL-tethered mRNA and reduce fertility. Therefore, PGL dimerization lies at the heart of both P-granule assembly and function. Finally, we identify the P-granule-associated Argonaute WAGO-1 as crucial for repression of PGL-tethered mRNA. We conclude that P-granule function requires both assembly and localized regulators.

developmental biology↗

bioRxiv2018

Brain folding is initiated by mechanical constraints without a cellular pre-pattern

During human brain development the cerebellum and cerebral cortex fold into robust patterns that increase and compartmentalize neural circuits. Although differential expansion of elastic materials has been proposed to explain brain folding, the cellular and physical processes responsible at the time of folding have not been defined. Here we used the murine cerebellum, with 8-10 folds, as a tractable model to study brain folding. At folding initiation we considered the cerebellum as a bilayer system with a fluidlike outer layer of proliferating precursors and an incompressible core. We discovered that there is no obvious cellular pre-pattern for folding, since when folding initiates, the precursors within the outer layer have uniform sizes, shapes and proliferation, as well as a distribution of glial fibers. Furthermore, although differential expansion is created by the outer layer expanding faster than the core at folding initiation, thickness variations arise in the outer layer that are inconsistent with elastic material models. A multiphase model was applied that includes radial and circumferential tension and mechanical constraints derived from in vivo measurements. Our results demonstrate that cerebellar folding emerges from mechanical forces generated by uniform cell behaviors. We discuss how our findings apply to human cerebral cortex folding.

developmental biology↗

bioRxiv2018

Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors

BackgroundThe cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known.\n\nMethodsWe used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atohl-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion.\n\nResultsMosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved.\n\nConclusionWe demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of conditional mutants affecting GCPs.

developmental biology↗

bioRxiv2018

Ankyrin domain encoding genes resulting from an ancient horizontal transfer are functionally integrated into developmental gene regulatory networks in the wasp Nasonia

BackgroundHow and why regulatory networks incorporate additional components, and how novel genes are maintained and functionally integrated into developmental processes are two important and intertwined questions whose answers have major implications for the evolution of development. We recently described a set of novel genes with robust and unique expression patterns along the dorsal-ventral axis of the embryo of the wasp Nasonia. Given the unique evolutionary history of these genes, and their apparent integration in to the dorsal-ventral (DV) patterning network, they are collectively an excellent model to study the evolution of regulatory networks, and the fates of novel genes.\n\nResultsWe have found that the novel DV genes are part of a large family of rapidly duplicating and diverging ankyrin domain encoding genes that originated most likely by horizontal transfer from Wolbachia in a common ancestor of the wasp superfamilly Chalcidoidea. We tested the function of those ankyrin encoding genes expressed along the DV axis and found that they participate in early embryonic DV patterning. We also developed a new wasp model system (Melittobia) and found that some functional integration of ankyrin genes have been preserved for over 90 million years, while others are lineage specific.\n\nConclusionsOur results indicate that regulatory networks can incorporate novel genes that then become necessary for stable and repeatable outputs. Even modest role in developmental networks may be enough to allow novel or duplicate genes to be maintained in the genome and become fully integrated network components.

developmental biology↗

bioRxiv2018

Transcriptomic and functional analysis of the oosome, a unique form of germplasm in the wasp Nasonia vitripennis

BackgroundThe oosome is the germline determinant in the wasp Nasonia vitripennis and is homologous to the polar granules of Drosophila. Despite a common evolutionary origin and developmental role, the oosome is morphologically quite distinct from polar granules. It is a solid sphere that migrates within the cytoplasm before budding out and forming pole cells.\n\nResultsTo gain an understanding of both the molecular basis of the novel form of the oosome, and the conserved essential features of germ plasm, we quantified and compared transcript levels between embryo fragments that contained the oosome, and those that did not. The identity of the localized transcripts indicated that Nasonia uses different molecules to carry out conserved germ plasm functions. In addition, functional testing of a sample of localized transcripts revealed potentially novel mechanisms of ribonucleoprotein assembly and pole cell cellularization in the wasp.\n\nConclusionsOur results demonstrate that numerous novel and unexpected molecules have been recruited in order produce the unique characteristics of the oosome and pole cell formation in Nasonia. This work will serve as the basis for further investigation into the patterns of germline determinant evolution among insects, the molecular basis of extreme morphology of ribonucleoproteins, and the incorporation of novel components into developmental networks.

developmental biology↗

bioRxiv2018

Single-cell RNA-seq reveals dynamic transcriptome profiling in human early neural differentiation

BackgroundInvestigating cell fate decision and subpopulation specification in the context of the neural lineage is fundamental to understanding neurogenesis and neurodegenerative diseases. The differentiation process of neural-tube-like rosettes in vitro is representative of neural tube structures, which are composed of radially organized, columnar epithelial cells and give rise to functional neural cells. However, the underlying regulatory network of cell fate commitment during early neural differentiation remains elusive.\n\nResultsIn this study, we investigated the genome-wide transcriptome profile of single cells from six consecutive reprogramming and neural differentiation time points and identified cellular subpopulations present at each differentiation stage. Based on the inferred reconstructed trajectory and the characteristics of subpopulations contributing the most towards commitment to the central nervous system (CNS) lineage at each stage during differentiation, we identified putative novel transcription factors in regulating neural differentiation. In addition, we dissected the dynamics of chromatin accessibility at the neural differentiation stages and revealed active c/s-regulatory elements for transcription factors known to have a key role in neural differentiation as well as for those that we suggest are also involved. Further, communication network analysis demonstrated that cellular interactions most frequently occurred among embryoid body (EB) stage and each cell subpopulation possessed a distinctive spectrum of ligands and receptors associated with neural differentiation which could reflect the identity of each subpopulation.\n\nConclusionsOur study provides a comprehensive and integrative study of the transcriptomics and epigenetics of human early neural differentiation, which paves the way for a deeper understanding of the regulatory mechanisms driving the differentiation of the neural lineage.

developmental biology↗

bioRxiv2018

Mathematical modeling supports fate restriction in neurogenic progenitors of the embryonic ventral spinal cord

In the developing neural tube in chicken and mammals, neural stem cells proliferate and differentiate according to a stereotyped spatio-temporal pattern. Several actors have been identified in the control of this process, from tissue-scale morphogens patterning (Shh, BMP) to intrinsic determinants in neural progenitor cells. In a previous study (Bonnet et al. eLife 7, 2018), we have shown that the CDC25B phosphatase promotes the transition from proliferation to differentiation in a cell-cycle independent fashion. In this study, we set up a mathematical model linking progenitor modes of division to the dynamics of progenitors and differentiated populations. Here, we build on this previous model to propose a complete dynamical picture of this process. We start from the standard model in which progenitors are homogeneous and can perform any type of divisions (proliferative division yielding two progenitors, asymmetric neurogenic divisions yielding one progenitor and one neuron, and terminal symmetric divisions yielding two neurons). We constraint this model using published data about mode of divisions and population dynamics of progenitors/neurons at different developmental stages (Saade et al. Cell Reports 4, 2013), and check the effect of CDC25B gain of function in this context. Next, we explore the scenarios in which progenitors population is actually split into two different pools, one of which composed of cells that have lost the capacity to perform proliferative divisions (fate restriction). We show that one such scenario appears relevant and calls for further identification of the alternative role of CDC25B in such a fate restriction.

developmental biology↗