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bioRxiv2016

Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages

Skeletal muscles arise from diverse embryonic origins, yet converge on common regulatory programs involving muscle regulatory factor (MRF)-family genes. Here, we compare the molecular basis of myogenesis in two separate muscle groups in the simple chordate Ciona: the atrial and oral siphon muscles. Here, we describe the ontogeny of OSM progenitors and characterize the clonal origins of OSM founders to compare mechanisms of OSM specification to what has been established for ASM. We determined that, as is the case in the ASM, Ebf and Tbx1/10 are both expressed and function upstream of Mrf in the OSM founder cells. However, regulatory relationships between Tbx1/10, Ebf and Mrf differ between the OSM and ASM lineages: while Tbx1/10, Ebf and Mrf form a linear cascade in the ASM, Ebf and Tbx1/10 are expressed in the inverse temporal order and are required together in order to activate Mrf in the OSM founder cells.

Developmental Biology↗

bioRxiv2016

foxc1a genetically interacts with ripply1 to regulate mesp-ba expression and somitogenesis in the zebrafish embryo

Somitogenesis is a fundamental segmentation process that forms the vertebrate body plan. A network of transcription factors is essential in establishing the spatial temporal order of this process. One such transcription factor is mesp-ba which has an important role in determining somite boundary formation. Its expression in somitogenesis is tightly regulated by the transcriptional activator Tbx6 and the repressor Ripply1 via a feedback regulatory network. Loss of foxc1a function in zebrafish leads to lack of anterior somite formation and reduced mesp-ba expression. Here we examine how foxc1a interacts with the tbx6-ripply1 network to regulate mesp-ba expression. In foxc1a morphants, anterior somites did not form at 12.5 hours post fertilization (hpf). At 22 hpf posterior somites formed, whereas anterior somites remained absent. In ripply1 morphants, no somites were observed at any time point. The expression of mesp-ba was reduced in the foxc1a morphants and expanded anteriorly in ripply1 morphants. The tbx6 expression domain was smaller and shifted anteriorly in the foxc1a morphants. Double knockdown of foxc1a and ripply1 resulted in absence of anterior somite formation while posterior somites did form, suggesting a partial rescue of the ripply1 phenotype. However, unlike the single foxc1a morphants, expression of mesp-ba was restored in the anterior PSM. Expression of tbx6 was expanded anteriorly in the double morphants. In conclusion, both foxc1a and ripply1 morphants displayed defects in somitogenesis, but their individual loss of function had opposing effects on mesp-ba expression. Loss of ripply1 appears to have rescued the mesp-ba expression in the foxc1a morphant, suggesting that intersection of these parallel regulatory mechanisms is required for normal mesp-ba expression and somite formation.

Developmental Biology↗

bioRxiv2016

Cellular aspects of gonadal atrophy in P-M hybrid dysgenesis

Gonadal atrophy is the most typical and dramatic manifestation of intraspecific hybrid dysgenesis syndrome leading to sterility of Drosophila melanogaster dysgenic progeny. The P-M system of hybrid dysgenesis is primarily associated with germ cell degeneration during the early stages of Drosophila development at elevated temperatures. In the present study, we have defined the phase of germ cell death as beginning at the end of embryogenesis immediately following gonad formation. The early stages of germ cell formation in dysgenic flies showed sensitivity to developmental temperature increases at any stage of the Drosophila life cycle including the imago. Analysis of germ cell reactions to hybrid dysgenesis induction revealed significant changes in subcellular structure, especially mitochondria, prior to germ cell breakdown. The mitochondrial pathology can be the reason for the activation of cell death pathways in dysgenic germ cells and lead to gonadal atrophy.\n\nSummaryWe investigated the hybrid dysgenesis in the context of germ cell development and observed the dysgenic effect is associated with dramatic pathological changes in structure of germ cell mitochondria and lead to massive and quick cell death. The temperature-dependent screening of germ cells developmental pattern in dysgenic background showed that these cells are susceptible to the hybrid dysgenesis at any drosophila life-cycle stage, including in the imago.

Developmental Biology↗

bioRxiv2016

Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide. RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos.\n\nWe found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

Developmental Biology↗

bioRxiv2016

Trophoblast survival signaling during human placentation requires HIF-induced transcription of HSP70

Survival of trophoblast cells in the low oxygen environment of human placentation requires metalloproteinase-mediated shedding of HBEGF and downstream signaling. A matrix metalloproteinase (MMP) antibody array and quantitative RT-PCR revealed upregulation of MMP2 post-transcriptionally in human first trimester HTR-8/SVneo trophoblast cells and placental explants exposed to 2% O2. Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. Hypoxia inducible factors, HIF1A and EPAS1 (HIF2A), accumulated at 2% O2, and HIF target genes were identified by next-generation sequencing of RNA from trophoblast cells cultured at 2% O2 for 0, 1, 2 and 4 hrs. Of nine genes containing HIF-response elements upregulated at 1 hour, only HSPA6 (HSP70B) remained elevated after 4 hours. The HSP70 chaperone inhibitor VER155008 blocked upregulation of both MMP2 and HBEGF at 2% O2, and increased apoptosis. However, both HBEGF upregulation and apoptosis were rescued by exogenous MMP2. We propose that MMP2-mediated shedding of HBEGF, initiated by HSP70, contributes to trophoblast survival at the low O2 levels encountered during the first trimester, and is essential for successful pregnancy outcomes.\n\nSummary StatementTrophoblast survival during human placentation, when oxygenation is minimal, required HIF-induced transcription of HSP70, which mediated MMP2 accumulation and the transactivation of anti-apoptotic ERBB signaling by HBEGF shedding.

Developmental Biology↗

bioRxiv2016

Divergence of gene regulatory network linkages during specification of ectoderm and mesoderm in early development of sea urchins

Developmental gene regulatory networks (dGRNs) are assemblages of interacting regulatory factors that direct ontogeny of animal body plans. The hierarchical topology of these networks predicts that their nodes will evolve at different rates and consequently will bias the trajectories of embryonic evolution. To test this, detailed, comparative analyses of dGRNs that specify early, global embryonic domains are required. The most extensively detailed dGRNs have been documented for one of the two subclasses of extant sea urchins, the euechinoids. Remarkably, euechinoid dGRNs operating in early development show little appreciable change even though they diverged approximately 90 million years ago (mya). Therefore, to better understand the evolutionary dynamics of dGRNs, comparative microdissection must be undertaken for sea urchins that diverged deeper in geological time. Recent studies of cidaroids, the sister clade of euechinoid sea urchins, suggest that comparative analyses of their embryonic domain specification may prove insightful for understanding the evolutionary dynamics of dGRNs. Here, I report the spatiotemporal dynamics of 19 regulatory factors involved in dorsal-ventral patterning of non-skeletogenic mesodermal and ectodermal domains in the early development of Eucidaris tribuloides, a cidaroid sea urchin. Multiple lines of evidence indicate that deployment of ectodermal regulatory factors is more impervious to change than mesodermal regulatory factors in the sea urchin lineage and are supported by multiple lines of experimental evidence. Additionally, endogenous spatiotemporal expression data, intra-class reporter microinjections, and perturbation analyses of Nodal and Notch signaling allow the enumeration of numerous alterations to regulatory factor deployment since the divergence of echinoids. These results provide a global view of early embryonic developmental processes in two clades that diverged at least 268.8 mya and show that the dGRNs controlling embryonic specification exhibit differential lability, supporting the hypothesis that the topologies of dGRNs bias rates of evolutionary change and alter the developmental evolutionary trajectories of embryogenesis.\n\nAuthor SummaryEarly in the development of an embryo, networks of genes are initiated to differentiate the rapidly dividing cells into distinct territories that will later serve specific functions. Sea urchins have revealed much about how this process unfolds. Recent studies have focused on one of the two modern lineages of sea urchins and have shown that these processes have not appreciably changed over the past 90 million years. I sought to determine if this trend extends over even larger evolutionary distances by investigating similar processes in a sea urchin from the second modern lineage, which is removed by 268 million years of evolution. By revealing where and when these genes are expressed and interfering with common mechanisms of development in a distantly related sea urchin, I show that changes to these networks of genes have occurred at all levels of the network. Additionally, I present data that suggests that changes to these networks of genes occur disproportionately in certain embryonic territories, which may be true for early development for other groups of organisms as well.

Developmental Biology↗

bioRxiv2016

Light sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development

BACKGROUNDSelective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratorys own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal.\n\nRESULTSOur OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm Maritigrella crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of Maritigrella with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope.\n\nCONCLUSIONSWe conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our gained experience during this project will help future OpenSPIM users with similar ambitions.

Developmental Biology↗

bioRxiv2016

Genotype-specific developmental plasticity shapes the timing and robustness of reproductive capacity in Caenorhabditis elegans

The effects of environmental stress on developmental phenotypes allows us to observe the adaptive capacity of various genotypes. In single-genotype populations, this allows for observing the effects of standing variation on the process of developmental plasticity. Typically, such efforts do not allow us to distinguish the effect of genotype from the effects of environment. In the Nematode Caenorhabditis elegans, however, the L1 stage of development allows for precise and controlled imposition of starvation on a synchronized population of individuals. This allows for systematic comparisons within and between defined genotypes.\n\nThis study provides a number of innovations in the study of Nematode developmental plasticity. The first is to demonstrate systems-level mechanisms of adaptive response for various genetic mutant phenotypes to extended L1 arrest. The effects of developmental deprivation are quantifiable by applying statistical modeling techniques to reproductive time-series. This is characterized for both a wildtype strain (N2) and a host of genetic mutant strains. A further contribution is investigating the effects of starvation at multi-generational timescales. This includes both two generations post-starvation and multiple starvation experiences with selection for fecundity over 11 generations.\n\nStatistical techniques such as Kernel Density Estimation (KDE), fold-change analysis, and Area Under the Curve (AUC) analysis reveal differences from baseline in the form of shifts in reproductive timing and kurtosis in peak reproductive capacity characterize adaptive responses that are semi-independent of environment. Extending these patterns of response to ad hoc comparisons with multi-generational contexts reveals that different genotypes characterized by mutations of specific genetic loci lead to vastly different outcomes.

Developmental Biology↗

bioRxiv2016

Transvection and pairing of a Drosophila Hox long noncoding RNA in the regulation of Sex combs reduced

Long noncoding RNAs have emerged as abundant and important regulators of gene expression in diverse animals. In D. melanogaster several lncRNAs involved in regulating Hox gene expression in the Bithorax Complex have been reported. However, no functional Hox long noncoding RNAs have been described in the Antennapedia Complex. Here we have characterized a long noncoding RNA lincX from the Antennapedia Complex, that is transcribed from previously identified cis-regulatory sequences of the Hox gene Sex combs reduced (Scr). We use both the GAL4-UAS system and mutants to ectopically overexpress the lincX RNA from exogenous and endogenous loci respectively, in order to dissect the potential regulatory functions of lincX RNA versus lincX transcription. Our findings suggest that transcription through the lincX locus, but not the lincX RNA itself, may facilitate initiation of Scr in cis in the early embryo. Transvection phenomena, where regulatory sequences on one chromosome can affect expression on the homolog, have previously been reported in genetic studies of Scr. By analysing lincX and Scr nascent transcriptional sites in embryos heterozygous for a gain of function mutation, we directly visualize this transvection, and observe that the ectopic lincX transcriptional state appears to be relayed in trans to the homologous wild-type chromosome. This trans-activation of lincX correlates with both ectopic activation of Scr in cis, and increased chromosomal proximity. Our results are consistent with a model whereby early long noncoding RNA transcription through cis-regulatory sequences can be communicated between chromosomes, and facilitates long-range initiation of Hox gene expression in cis.

Developmental Biology↗

bioRxiv2016

Morphological diversity of blastula formation and gastrulation in temnopleurid sea urchins

Embryos of temnopleurid sea urchins exhibit species-specific morphologies. While Temnopleurus toreumaticus has a wrinkled blastula, others have a smooth blastula. Embryos of T. toreumaticus invaginate continuously at gastrulation, whereas in some others invagination is stepwise. We studied blastula and gastrula formation in four temnopleurids using light and scanning electron microscopy to clarify the mechanisms producing these differences. Unlike T. toreumaticus, blastomeres of mid-blastulae in T. reevesii, T. hardwickii and Mespilia globulus formed pseudopods. Before primary mesenchyme cells ingressed, embryos developed an area of orbicular cells in the vegetal plate. The cells surrounding the orbicular cells extended pseudopods toward the orbicular cell area in T. toreumaticus, T. reevesii and T. hardwickii. In T. toreumaticus, the extracellular matrix was well-developed and developed a hole-like structure that was not formed in others. Gastrulation of T. reevesii, T. hardwickii and M. globulus was stepwise, suggesting that differences of gastrulation are caused by all or some of factors: change of cell shape, rearrangement, pushing up and towing of cells. These species-specific morphologies may be caused by the shape and surface structure of blastomeres with cell-movement.\n\nSummary statementTemonopleurid embryology

Developmental Biology↗

bioRxiv2016

A Transcriptional Lineage of the Early C. elegans Embryo

HIGHLIGHTS- RNA-seq on each cell of the early C. elegans embryo complements the known lineage\n- We measured the zygotic activation specific to each unique cell of the embryo\n- We identified genes that are functionally redundant and critical for development\n- We created an interactive online data visualization tool for exploring the data\n\n\neTOC BLURBC. elegans is a powerful model for development, with an invariant and completely described cell lineage. To enrich this resource, we performed single-cell RNA-seq on each cell of the embryo through the 16-cell stage. Zygotic genome activation is differential between cell types. We identified hundreds of candidates for partially redundant genes, and verified one such set as critical for development. We created an interactive online data visualization tool to invite others to explore our dataset.\n\nSUMMARYDuring embryonic development, cells must establish fates, morphologies and behaviors in coordination with one another to form a functional body. A prevalent hypothesis for how this coordination is achieved is that each cells fate and behavior is determined by a defined mixture of RNAs. Only recently has it become possible to measure the full suite of transcripts in a single cell. Here we quantify the abundance of every mRNA transcript in each cell of the C. elegans embryo up to the 16-cell stage. We describe spatially dynamic expression, quantify cell-specific differential activation of the zygotic genome, and identify critical developmental genes previously unappreciated because of their partial redundancy. We present an interactive data visualization tool that allows broad access to our dataset. This genome-wide single-cell map of mRNA abundance, alongside the well-studied life history and fates of each cell, describes at a cellular resolution the mRNA landscape that guides development.

Developmental Biology↗

bioRxiv2016

Live Imaging of muscle histolysis in Drosophila metamorphosis

BackgroundThe contribution of programmed cell death (PCD) to muscle wasting disorders remains a matter of debate. Drosophila melanogaster metamorphosis offers the opportunity to study muscle cell death in the context of development. Using live cell imaging of the abdomen, two groups of larval muscles can be observed, doomed muscles that undergo histolysis and persistent muscles that are remodelled and survive into adulthood.\n\nMethodTo identify and characterize genes that control the decision between survival and cell death of muscles, we developed a method comprising in vivo imaging, targeted gene perturbation and time-lapse image analysis. Our approach enabled us to study the cytological and temporal aspects of abnormal cell death phenotypes.\n\nResultsIn a previous genetic screen for genes controlling muscle size and cell death in metamorphosis, we identified gene perturbations that induced cell death of persistent or inhibit histolysis of doomed larval muscles. RNA interference (RNAi) of the genes encoding the helicase Rm62 and the lysosomal Cathepsin-L homolog Cysteine proteinase 1 (Cp1) caused premature cell death of persistent muscle in early and mid-pupation, respectively. Silencing of the transcriptional co-repressor Atrophin inhibited histolysis of doomed muscles. Overexpression of dominant-negative Target of Rapamycin (TOR) delayed the histolysis of a subset of doomed and induced ablation of all persistent muscles. RNAi of AMPK, which encodes a subunit of the AMPK protein complex that senses AMP and promotes ATP formation, led to loss of attachment and a spherical morphology. None of the perturbations affected the survival of newly formed adult muscles, suggesting that the method is useful to find genes that are crucial for the survival of metabolically challenged muscles, like those undergoing atrophy. The ablation of persistent muscles did not affect eclosion of adult flies.\n\nConclusionsLive imaging is a versatile approach to uncover gene functions that are required for the survival of muscle undergoing remodelling, yet are dispensable for other adult muscles. Our approach promises to identify molecular mechanisms that can explain the resilience of muscles to PCD.

Developmental Biology↗

bioRxiv2016

Guided self-organization recapitulates tissue architecture in a bioengineered brain organoid model

Recently emerging methodology for generating human tissues in vitro has the potential to revolutionize drug discovery and disease research. Currently, three-dimensional cell culture models either rely on the pronounced ability of mammalian cells to self organize in vitro1-6, or use bioengineered constructs to arrange cells in an organ-like configuration7,8. While self-organizing organoids can recapitulate developmental events at a remarkable level of detail, bioengineered constructs excel at reproducibly generating tissue of a desired architecture. Here, we combine these two approaches to reproducibly generate micropatterned human forebrain tissue while maintaining its self-organizing capacity. We utilize poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a scaffold to generate elongated embryoid bodies and demonstrate that this influences tissue identity. Micropatterned engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, we reconstitute the basement membrane at later stages leading to characteristic cortical tissue architecture including formation of a polarized cortical plate and radial units. enCORs provide the first in vitro system for modelling the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. We demonstrate their utility by modelling teratogenic effects of ethanol and show that defects in leading process formation may be responsible for the neuronal migration deficits in fetal alcohol syndrome. Our data demonstrate that combining 3D cell culture with bioengineering can significantly enhance tissue identity and architecture, and establish organoid models for teratogenic compounds.

Developmental Biology↗

bioRxiv2016

Activated Ras expression in eye discs with altered levels of hsrω lncRNA causes JNK-induced Dilp8 secretion and reduces post-pupal ecdysone leading to early pupal death in Drosophila

BackgroundDilp8-mediated inhibition of ecdysone synthesis and pupation in holometabolous insects maintains developmental homeostasis through stringent control of timing and strength of moulting signals. We examined reasons for normal pupation but early pupal death observed in certain cases.\n\nResultsOver-expression of activated Ras in developing eye/wing discs inhibited Ptth expression in brain via up-regulated JNK signaling mediated Dilp8 secretion from imaginal discs, which inhibited ecdysone synthesis in prothoracic gland after pupariation, leading to death of ~25-30 Hr old pupae. Inhibition of elevated Ras signaling completely rescued early pupal death while post-pupation administration of ecdysone to organisms with elevated Ras signaling in eye discs partially rescued their early pupal death. Unlike the earlier known Dilp8 action in delaying pupation, hyperactivated Ras mediated elevation of pJNK signaling in imaginal discs caused Dilp8 secretion after pupariation. Ectopic expression of certain other transgene causing pupal lethality similarly enhanced pJNK and early pupal Dilp8. Sub-optimal ecdysone levels after 8 Hr of pupation prevented the early pupal metamorphic changes and caused organismal death.\n\nConclusionsOur results reveal early pupal stage as a novel Dilp8 mediated post-pupariation checkpoint and provide further evidence for inter-organ signaling during development, wherein a peripheral tissue influences the CNS driven endocrine function.

Developmental Biology↗

bioRxiv2016

Dual functions of Intraflagellar Transport Protein IFT20 in spermiogenesis: formation of sperm flagella and removal of cytoplasm by autophagy

Intraflagellar transport (IFT) is a conserved mechanism thought to be essential for the assembly and maintenance of cilia and flagella. However, little is known about mammalian sperm flagella formation. To fill this gap, we disrupted the Ift20 gene in male germ cells. Homozygous mutant mice were infertile with significantly reduced sperm counts and motility. In addition, abnormally shaped elongating spermatid heads and bulbous round spermatids were found in the lumen of the seminiferous tubules. Electron microscopy revealed increased cytoplasmic vesicles, fiber-like structures, abnormal accumulation of mitochondria and decreased mature lysosomes. The few developed sperm had disrupted axoneme and retained cytoplasmic lobe components on the flagella. ODF2 and SPAG16L, two sperm flagella proteins failed to be incorporated into sperm tails of the mutant mice. Expression levels of an autophagy core protein that associates with IFT20, Atg16, were significantly reduced in the testis of the Ift20 mutant mice. Our studies suggest that IFT20 is essential for spermiogenesis in mice, and it plays a role in sperm flagella formation, and removing excess cytoplasmic components by regulating autophagy core proteins.

Developmental Biology↗

bioRxiv2016

Interactions between Nodal and Wnt signalling Drive Robust Symmetry Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)

Generation of asymmetry within the early embryo is a critical step in the establishment of the three body axes, providing a reference for the patterning of the organism. To study the establishment of asymmetry and the development of the anteroposterior axis (AP) in culture, we utilised our Gastruloid model system. Gastruloids, highly reproducible embryonic organoids formed from aggregates of mouse embryonic stem cells, display symmetry-breaking, polarised gene expression and axial development, mirroring the processes on a time-scale similar to that of the mouse embyro. Using Gastruloids formed from mouse ESCs containing reporters for Wnt, FGF and Nodal signalling, we were able to quantitatively assess the contribution of these signalling pathways to the establishment of asymmetry through single time-point and live-cell fluorescence microscopy.\n\nDuring the first 24-48h of culture, interactions between the Wnt/{beta}-Catenin and Nodal/TGF{beta} signalling pathways promote the initial symmetry-breaking event, manifested through polarised Brachyury (T/Bra) expression. Neither BMP nor FGF signalling is required for the establishment of asymmetry, however Wnt signalling is essential for the amplification and stability of the initial patterning event. Additionally, low, endogenous levels of FGF (24-48h) has a role in the amplification of the established pattern at later time-points.\n\nOur results confirm that Gastruloids behave like epiblast cells in the embryo, leading us to translate the processes and signalling involved in pattern formation of Gastruloids in culture to the development of the embryo, firmly establishing Gastruloids as a highly reproducible, robust model system for studying cell fate decisions and early pattern formation in culture.

Developmental Biology↗

bioRxiv2016

Odd-paired controls frequency doubling in Drosophila segmentation by altering the pair-rule gene regulatory network

The Drosophila embryo transiently exhibits a double segment periodicity, defined by the expression of seven \"pair-rule\" genes, each in a pattern of seven stripes. At gastrulation, interactions between the pair-rule genes lead to frequency doubling and the patterning of fourteen parasegment boundaries. In contrast to earlier stages of Drosophila anteroposterior patterning, this transition is not well understood. By carefully analysing the spatiotemporal dynamics of pair-rule gene expression, we demonstrate that frequency-doubling is precipitated by multiple coordinated changes to the network of regulatory interactions between the pair-rule genes. We identify the broadly expressed but temporally patterned transcription factor, Odd-paired (Opa/Zic), as the cause of these changes, and show that the patterning of the even-numbered parasegment boundaries relies on Opa-dependent regulatory interactions. Our findings indicate that the pair-rule gene regulatory network has a temporally-modulated topology, permitting the pair-rule genes to play stage-specific patterning roles.

Developmental Biology↗

bioRxiv2016

Dynamic changes in Sox2 spatio-temporal expression direct the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos

Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4 and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerise on adjacent cis regulatory elements, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In this study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy (FCS). We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4s Sox/Oct motif. Taking into account that Sox2 expression levels fluctuate more than Oct4, our finding could explain how Sox2 controls the segregation of the cells of the inner cell mass (ICM) into the epiblast (EPI) and the primitive endoderm (PE) populations within the developing rodent blastocyst.

Developmental Biology↗