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bioRxiv2014

Systematic Imaging Reveals Features of Localized mRNAs and Their Changing Subcellular Destinations in Development

mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary, depends on the microtubule cytoskeleton and the mRNAs enriched at the posterior cortex share common localization machinery. Localized mRNAs, in particular the posterior class, have distinct gene features and differ in expression level, 3UTR length and sequence conservation from unlocalized mRNAs. Using cross-tissue comparison we revealed that the localization status of mRNAs differs between epithelial, germline and embryonic cell types and also changes within one cell, the oocyte, over time. This dataset enables the transition from deep mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

Developmental Biology↗

bioRxiv2014

Epithelial flow into the optic cup facilitated by suppression of BMP drives eye morphogenesis

The transformation of the oval optic vesicle to a hemispheric bi-layered optic cup involves major morphological changes during early vertebrate eye development. According to the classical view, the lens-averted epithelium differentiates into the retinal pigmented epithelium (RPE), while the lens-facing epithelium forms the neuroretina. We find a 4.7 fold increase of the entire basal surface of the optic cup.\n\nAlthough the area an individual RPC demands at its basal surface declines during optic cup formation, we find a 4.7 fold increase of the entire basal surface of the optic cup. We demonstrate that the lens-averted epithelium functions as reservoir and contributes to the growing neuroretina by epithelial flow around the distal rims of the optic cup. This flow is negatively modulated by BMP, which arrests epithelial flow. This inhibition results in persisting neuroretina in the RPE domain and ultimately in coloboma.

Developmental Biology↗

bioRxiv2014

Interplay of TGFb superfamily members governs optic fissure closure

The optic fissure is a gap in the developing vertebrate eye and must be closed as development proceeds. A persisting optic fissure is referred to as coloboma, a major cause for blindness in children. Multiple factors have been linked to coloboma formation, however, the actual process of fissure closure is only poorly understood.\n\nBased on our findings we propose an important role of TGFb signaling for optic fissure closure. We show active TGFb signaling in the fissure margins, analyzed by a new TGFb signaling reporter zebrafish. We found BMP antagonists regulated by TGFb. These antagonists we also found expressed in the fissure margins. Finally we show a coloboma phenotype in a TGFb KO mouse. Microarray data analysis indicates intense TGFb dependent remodeling of the extracellular matrix (ECM) during optic fissure closure.\n\nWe propose that TGFb is driving optic fissure closure by ECM remodeling. As previously shown, inhibition of BMP signaling is important for such TGFb dependent ECM remodeling. We show that this is achieved by the regulation of BMP antagonists, expressed in the optic fissure margins.

Developmental Biology↗

bioRxiv2014

The role of TORC1 in muscle development in Drosophila.

Myogenesis is an important process during both development and muscle repair. Previous studies suggest that mTORC1 plays a role in the formation of mature muscle from immature muscle precursor cells. Here we show that gene expression for several myogenic transcription factors including Myf5, Myog and Mef2c but not MyoD and myosin heavy chain isoforms decrease when C2C12 cells are treated with rapamycin, supporting a role for mTORC1 pathway during muscle development. To investigate the possibility that mTORC1 can regulate muscle in vivo we ablated the essential dTORC1 subunit Raptor in Drosophila melanogaster and found that muscle-specific knockdown of Raptor causes flies to be too weak to emerge from their pupal cases during eclosion. Using a series of GAL4 drivers we also show that muscle-specific Raptor knockdown also causes shortened lifespan, even when eclosure is unaffected. Together these results highlight an important role for TORC1 in muscle development, integrity and function in both Drosophila and mammalian cells.

Developmental Biology↗

bioRxiv2014

Genome-wide Ultrabithorax binding analysis reveals highly targeted genomic loci at developmental regulators and a potential connection to Polycomb-mediated regulation

Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax (Ubx) directs the development of thoracic and abdominal segments and appendages, and loss of Ubx function can lead for example to the transformation of third thoracic segment appendages (e.g. halters) into second thoracic segment appendages (e.g. wings), resulting in a characteristic four-wing phenotype. Here we present a Drosophila melanogaster strain with a V5-epitope tagged Ubx allele, which we employed to obtain a high quality genome-wide map of Ubx binding sites using ChIP-seq. We confirm the sensitivity of the V5 ChIP-seq by recovering 7/8 of well-studied Ubx-dependent cis-regulatory regions. Moreover, we show that Ubx binding is predictive of enhancer activity as suggested by comparison with a genome-scale resource of in vivo tested enhancer candidates. We observed densely clustered Ubx binding sites at 12 extended genomic loci that included ANTP-C, BX-C, Polycomb complex genes, and other regulators and the clustered binding sites were frequently active enhancers. Furthermore, Ubx binding was detected at known Polycomb response elements (PREs) and was associated with significant enrichments of Pc and Pho ChIP signals in contrast to binding sites of other developmental TFs. Together, our results show that Ubx targets developmental regulators via strongly clustered binding sites and allow us to hypothesize that regulation by Ubx might involve Polycomb group proteins to maintain specific regulatory states in cooperative or mutually exclusive fashion, an attractive model that combines two groups of proteins with prominent gene regulatory roles during animal development.

Developmental Biology↗

bioRxiv2015

Digit evolution in gymnophthalmid lizards

BackgroundThe tetrapod limb is a highly diverse structure, and reduction of the limbs accounts for much of the phenotypes observed within species. Squamate reptiles represent one of the many lineages in which the limbs have been greatly modified from the pentadactyl generalized pattern; within the group, limb-reduced morphologies can vary from minor reductions in size of elements to complete limblessness, with several intermediate forms in between. Even though limb reduction is widespread, it is not clear what are the evolutionary and developmental mechanisms involved in the formation of reduced limb morphologies.\n\nMethodsIn this study, we present an overview of limb morphology within the microteiid lizard group Gymnophthalmidae, focusing on digit number.\n\nResultsWe show that there are two major groups of limb-reduced gymnophthalmids. The first group is formed by lizard-like - and frequently pentadactyl - species, in which minor reductions (such as the loss of 1-2 phalanges mainly in digits I and V) are the rule; these morphologies generally correspond to those seen in other squamates. The second group is formed by species showing more drastic losses, which can include the absence of an externally distinct limb in adults. We also show the expression patterns of Sonic Hedgehog (Shh) in the greatly reduced fore and hindlimb of a serpentiform gymnophthalmid.\n\nConclusionsOur discussion focus on identifying shared patterns of limb reduction among tetrapods, and explaining these patterns and the morphological variation within the gymnophthalmids based on the current knowledge of the molecular signaling pathways that coordinate limb development.

Developmental Biology↗

bioRxiv2015

Mbd3/NuRD is a Key Inhibitory Module During the Induction and Maintenance of Naïve Pluripotency

Our group has published a study on induced pluripotent stem cell (iPSC) reprogramming (Rais et al. Nature 20131) that reached the following conclusions: a) Mbd3/NuRD is a repressor of inducing naive pluripotency from mouse Epiblast stem cells (EpiSCs), primordial germ cells (PGCs), murine somatic cells and human secondary fibroblasts; b) Up to 100% iPSC formation efficiency can be achieved via optimized Mbd3/NuRD depletion, in concert with optimized OKSM delivery and naive pluripotency conditions (2i supplement applied only after 48 hours, human LIF, hypoxia and Vitamin C containing Knockout serum replacement)1. This represented the first proof for deterministic/near-deterministic iPSC reprogramming, and highlighted a previously unappreciated role for Mbd3/NuRD in hampering the re-establishment of pluripotency. Recent reports have seemingly provided contradictory results and attempted to dispute our iPSC efficiency quantifications and/or the role of Mbd3/NuRD in blocking reprogramming2,3. Here we provide a detailed response to these reports based on extended discussions and providing new data. The synthesis presented herein disagrees with claims made by Silva, Hendrich, Bertone and colleagues2,3, and reconfirms that Mbd3/NuRD is a major pathway that inhibits the maintenance and induction of pluripotency1. Further, we foresee that its controlled manipulation is likely to become an integral pathway for inducing and maintaining naive pluripotency in a variety of species.

Developmental Biology↗

bioRxiv2015

FGF/MAPK signaling sets the switching threshold of a bistable circuit controlling cell fate decisions in ES cells

Intracellular transcriptional regulators and extracellular signaling pathways together regulate the allocation of cell fates during development, but how their molecular activities are integrated to establish the correct proportions of cells with particular fates is not known. Here we study this question in the context of the decision between the epiblast (Epi) and the primitive endoderm (PrE) fate that occurs in the mammalian preimplantation embryo. Using an embryonic stem (ES) cell model, we discover two successive functions of FGF/MAPK signaling in this decision. First, the pathway needs to be inhibited to make the PrE-like gene expression program accessible for activation by GATA transcription factors in ES cells. In a second step, MAPK signaling levels determine the threshold concentration of GATA factors required for PrE-like differentiation, and thereby control the proportion of cells differentiating along this lineage. Our findings can be explained by a simple mutual repression circuit modulated by FGF/MAPK signaling. This may be a general network architecture to integrate the activity of signal transduction pathways and transcriptional regulators, and serve to balance proportions of cell fates in several contexts.

Developmental Biology↗

bioRxiv2015

Nitric oxide regulates growth coordination during regeneration

Mechanisms that coordinate the growth of different tissues during development are essential for producing adult animals with proper organ proportion. Here we describe a pathway through which tissues communicate with each other to coordinate growth. During Drosophila melanogaster larval development, damage to imaginal discs activates a regeneration checkpoint that produces both a delay in developmental timing and slows the growth of undamaged tissues, coordinating regeneration of the damaged tissue with developmental progression and overall growth. Both developmental delay and growth control are mediated by secretion of the insulin/relaxin family peptide Dilp8 from regenerating tissues. Here we demonstrate that Dilp8-dependent growth coordination between regenerating and undamaged tissues, but not developmental delay, requires the activity of nitric oxide synthase (NOS) in the prothoracic gland. NOS limits the growth of undamaged tissues by reducing ecdysone biosynthesis, a requirement for imaginal disc growth during both the regenerative checkpoint and normal development. Therefore, NOS activity in the prothoracic gland translates information about the growth status of individual tissues into coordinated tissue growth through the regulation of endocrine signals.

Developmental Biology↗

bioRxiv2015

Tissue dynamics of the forebrain neural plate

The forebrain has the most complex shape and structure of the vertebrate brain regions and the mechanisms of its formation remain obscure. Convergence and extension movements are characteristic of the posterior (spinal cord and hindbrain) neural plate (pNP) while tissue deformations and underlying cellular dynamics during the early shaping of the forebrain neural plate (fNP) are undefined. Here, we apply live imaging, automated cell tracking and computational analysis to quantitatively map cell behaviour in the zebrafish fNP. We demonstrate a novel mechanism in which planar cell rearrangements, with a passive signature, are orthogonal to those in the pNP, and cell divisions lacking planar-polarity facilitate thickening from two to three layers. We develop a mechanical model of the fNP in which polarised cell behaviour arises from interactions with dissimilar bordering tissues rather than from intrinsically polarised cells. The model unifies in vivo observations and provides a mechanistic understanding of fNP morphogenesis.

Developmental Biology↗

bioRxiv2015

A general theory of individuated multicellularity

Changes in gene expression are thought to regulate the differentiation process intrinsically through complex epigenetic mechanisms. In fundamental terms, however, this assumed regulation refers only to the intricate propagation of changes in gene expression or else leads to logical inconsistencies. The evolution and self-regulatory dynamics of individuated multicellularity also lack a unified and falsifiable description. To fill this gap, I computationally analyzed publicly available high-throughput data of histone H3 post-translational modifications and mRNA abundance for different Homo sapiens, Mus musculus, and Drosophila melanogaster cell-type/developmental-periods samples. My analysis of genomic regions adjacent to transcription start sites generated a profile from pairwise partial correlations between histone modifications controlling for the respective mRNA levels for each cell-type/developmental-period dataset. I found that these profiles, while explicitly uncorrelated to transcript abundance by construction, associate strongly with cell differentiation states. This association is not expected if cell differentiation is, in effect, regulated by epigenetic mechanisms. Based on these results, I propose a falsifiable theory of individuated multicellularity, which relies on the synergistic coupling across the extracellular space of two stochastically independent \"self-organizing\" systems constraining histone modification states at the same sites. This theory describes how the multicellular individual--understood as an intrinsic, higher-order constraint--emerges from proliferating undifferentiated cells, and may explain the intrinsic regulation of gene transcriptional changes during cell differentiation and the evolution of individuated multicellular organisms.

Developmental Biology↗

bioRxiv2015

The unusual gene order in the Echinoderm Hox cluster is related to the embryo and larva symmetries

BackgroundHox gene collinearity relates the sequential location of Hox genes in the 3 to 5 direction on the chromosome with the linear arrangement of the body elements along the anterior-posterior (A/P) axis of bilaterian embryos. This spatial Hox gene collinearity has been almost universally respected in diverse organisms like worms, insects or vertebrates. It is therefore surprising that the above well established collinearity rule is violated in the case of Echinoderms. No explanation of this violation is apparent. Here a hypothesis is put forward which provides a cue to understand the abnormal serial gene location in the sea urchin disorganized Hox cluster.\n\nResultsBilateral symmetry along the A/P embryo axis is established at the very early stages of ontogeny of the sea urchin. For the subsequent developmental stages, rotational symmetry emerges in the vestibula larva. In analogy to the linear A/P case, the circular topology of modules might be a reflection of the architectural restructuring of the Hox loci where the 3 and 5 ends of the Hox cluster approach each other so that a closed contour of the chromatin fiber is formed. At a later stage, the break and opening of the cluster contour at the level of Hox4 combined with the rotational symmetry leads to the observed Hox gene sequence that violates the standard 3 to 5 collinearity.\n\nConclusionThe unusual gene series manifests the congruence of Hox gene sequence in the cluster with the circular arrangement of the sea urchin primary podia. Accordingly, the Hox sequence after the break at Hox4 is not a violation but an extension of Hox gene collinearity to animals with rotational symmetry.

Developmental Biology↗

bioRxiv2015

Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here, we mutated an invertebrate orphan relaxin receptor, the Drosophila Lgr3, and found body asymmetries similar to those found in insulin/relaxin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNAi against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By fluorescently-tagging the endogenous Lgr3 protein and performing CNS-specific RNAi, we find that Lgr3 is expressed and required in a novel subset of CNS neurons to transmit the peripheral tissue stress signal, Dilp8, to the neuroendocrine centers controlling developmental timing. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.

Developmental Biology↗

bioRxiv2015

Tribolium castaneum as a model for microRNA evolution, expression and function during short germband development

MicroRNAs are well-established players in the development of multicellular animals. Most of our understanding of microRNA function in arthropod development comes from studies in Drosophila. Despite their advantages as model systems, the long germband embryogenesis of fruit flies is an evolutionary derived state restricted to several holometabolous insect lineages. MicroRNA evolution and expression across development in animals exhibiting the ancestral and more widespread short germband mode of embryogenesis has not been characterized. We sequenced small RNA libraries of oocytes and successive time intervals covering the embryonic development of the short germband model organism, the red flour beetle Tribolium castaneum. We analysed the evolution and temporal expression of the microRNA complement, and sequenced libraries of total RNA to investigate the relationships with microRNA target expression. We show microRNA maternal loading and sequence-specific 3-end non-template oligoadenylation of maternally deposited microRNAs that is conserved between Tribolium and Drosophila. We further uncover large clusters encoding multiple paralogs from several Tribolium-specific microRNA families expressed during a narrow interval of time immediately after the activation of zygotic transcription. These novel microRNAs, together with several early expressed conserved microRNAs, target a significant number of maternally deposited transcripts. Comparison with Drosophila shows that microRNA-mediated maternal transcript targeting is a conserved phenomenon in insects, but the number and sequences of microRNAs involved in this process have diverged. The expression of fast-evolving and species-specific microRNAs in the early blastoderm of T. castaneum is consistent with previous findings in Drosophila and shows that the unique permissiveness for microRNA innovation at this stage is a conserved phenomenon.

Developmental Biology↗

bioRxiv2015

Heterogeneous generation of new cells in the adult echinoderm nervous system

Adult neurogenesis, generation of new functional cells in the mature central nervous system (CNS), has been documented in a number of diverse organisms, ranging from humans to invertebrates. However, the origin and evolution of this phenomenon is still poorly understood for many of the key phylogenetic groups. Echinoderms are one such phylum, positioned as a sister group to chordates within the monophyletic clade Deuterostomia. They are well known for the ability of their adult organs, including the CNS, to completely regenerate after injury. Nothing is known, however, about production of new cells in the nervous tissue under normal physiological conditions in these animals. In this study, we show that new cells are continuously generated in the mature radial nerve cord (RNC) of the sea cucumber Holothuria glaberrima. Importantly, this neurogenic activity is not evenly distributed, but is significantly more extensive in the lateral regions of the RNC than along the midline. Some of the new cells generated in the apical region of the ectoneural neuroepithelium leave their place of origin and migrate basally to populate the neural parenchyma. Gene expression analysis showed that generation of new cells in the adult sea cucumber CNS is associated with transcriptional activity of genes known to be involved in regulation of various aspects of neurogenesis in other animals. Further analysis of one of those genes, the transcription factor Myc showed that it is expressed, in some, but not all radial glial cells, suggesting heterogeneity of this CNS progenitor cell population in echinoderms.

Developmental Biology↗

bioRxiv2015

The relationship between interpregnancy interval (IPI) and mother’s preceding pregnancies (PP)

IntroductionFollowing the investigation of the mothers preceding pregnancies on fetal development and postnatal survival of the neonate, we turned our attention to an earlier period, that is the interval separating the onset of the current pregnancy from the end of the preceding one. The objectives of this study is to investigate the variations of interpregnancy interval length associated to the mothers preceding pregnancies.\n\nMethodsA population of 7773 neonates, alive at the time of hospital discharge, were divided into cohorts according to the current neonates sex and number and sex of the mothers preceding pregnancies. Interpregnancy interval average of each cohort of same neonates sex and mothers parity, but different configuration of preceding pregnancies, were measured and compared.\n\nResultsA positive association was found between mothers preceding pregnancies and length of interpregnancy interval when current pregnancy and preceding pregnancy were of the same sex, and a negative association when they were of opposite sex.\n\nDiscussionInterpregnancy interval length follows a pattern regarding the gravidas preceding pregnancy similar to the other early life indicators pattern, birth weight, placenta weight, gestation length and neonatal survival. Our results confirm and complete an immunological explanation of the indicators variations associated to the gravidas preceding pregnancy.

Developmental Biology↗

bioRxiv2015

The C. elegans ephrin EFN-4 functions non-cell autonomously with heparan sulfate proteoglycans to promote axon outgrowth and branching

The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body wall muscle, and in parallel with HS biosynthesis genes and HSPG core proteins, which function cell autonomously in the AIY neurons. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system.

Developmental Biology↗

bioRxiv2015

Capabilities and Limitations of Tissue Size Control Through Passive Mechanical Forces

Embryogenesis is an extraordinarily robust process, exhibiting the ability to control tissue size and repair patterning defects in the face of environmental and genetic perturbations. The size and shape of a developing tissue is a function of the number and size of its constituent cells as well as their geometric packing. How these cellular properties are coordinated at the tissue level to ensure developmental robustness remains a mystery; understanding this process requires studying multiple concurrent processes that make up morphogenesis, including the spatial patterning of cell fates and apoptosis, as well as cell intercalations. In this work, we develop a computational model that aims to understand aspects of the robust pattern repair mechanisms of the Drosophila embryonic epidermal tissues. Size control in this system has previously been shown to rely on the regulation of apoptosis rather than proliferation; however, to date little work has been done to understand the role of cellular mechanics in this process. We employ a vertex model of an embryonic segment to test hypotheses about the emergence of this size control. Comparing the model to previously published data across wild type and genetic perturbations, we show that passive mechanical forces suffice to explain the observed size control in the posterior (P) compartment of a segment. However, observed asymmetries in cell death frequencies across the segment are demonstrated to require patterning of cellular properties in the model. Finally, we show that distinct forms of mechanical regulation in the model may be distinguished by differences in cell shapes in the P compartment, as quantified through experimentally accessible summary statistics, as well as by the tissue recoil after laser ablation experiments.\n\nAuthor SummaryDeveloping embryos are able to grow organs of the correct size even in the face of significant external perturbations. Such robust size control is achieved via tissue-level coordination of cell growth, proliferation, death and rearrangement, through mechanisms that are not well understood. Here, we employ computational modelling to test hypotheses of size control in the developing fruit fly. Segments in the surface tissues of the fruit fly embryo have been shown to achieve the same size even if the number of cells in each segment is perturbed genetically. We show that simple mechanical interactions between the cells of this tissue can recapitulate previously gathered data on tissue sizes and cell numbers. However, this simple model does not capture the experimentally observed spatial variation in cell death rates in this tissue, which may be explained through several adaptations to the model. These distinct adaptations may be distinguished through summary statistics of the tissue behaviour, such as statistics of cell shapes or tissue recoil after cutting. This work demonstrates how computational modelling can help investigate the complex mechanical interactions underlying tissue size and shape, which are important for understanding the underlying causes of birth defects and diseases driven by uncontrolled growth.

Developmental Biology↗