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Results for “Developmental Biology”

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bioRxiv2018

Senescence of multicellular individuals: imbalance of epigenetic and non-epigenetic information in histone modifications

Cellular aging has been progressively elucidated by science. However, the fundamental cause of senescence--i.e., why organisms age at the multicellular-individual level--remains unclear. A recent theory of individuated multicellularity describes the emergence and growth of crucial information content for cell differentiation. This information is mostly conveyed in the non-epigenetic (i.e., transcription uncorrelated) histone crosstalk near transcription start sites. According to this theory, the non-epigenetic content emerges and grows at the expense of the information capacity for epigenetic content. If this "reassignment" of information capacity continues after adulthood, it may explain the senescence phenomenon. Here, I present a novel, falsifiable theory describing an uninterrupted growth of capacity for non-epigenetic information at the expense of that for epigenetic information not only during ontogeny but also throughout adulthood. As a byproduct, this continuous "reassignment" of capacity effectively creates an information imbalance in histone crosstalk, which in turn overregulates transcriptional levels. This overregulation is to be understood as transcriptional levels becoming more and more accurate but also less and less precise with respect to the needs of the multicellular individual--up to the point of dysfunctionality. This epigenetic/non-epigenetic information imbalance is proposed to be the primary reason why individuated multicellular organisms senesce.

developmental biology↗

bioRxiv2018

Cerebellar nuclei neurons dictate growth of the cortex through developmental scaling of presynaptic Purkinje cells

Efficient function of neural systems requires the production of specific cell types in the correct proportions. Here we report that reduction of the earliest born neurons of the cerebellum, excitatory cerebellar nuclei neurons (eCN), results in a subsequent reduction in growth of the cerebellar cortex due to an accompanying loss of their presynaptic target Purkinje cells. Conditional knockout of the homeobox genes En1 and En2 (En1/2) in the rhombic lip-derived eCN and granule cell precursors leads to embryonic loss of a subset of medial eCN and cell non-autonomous and location specific loss of Purkinje cells, with subsequent proportional scaling down of cortex growth. We propose that subsets of eCN dictate the survival of their specific Purkinje cell partners, and in turn sonic hedgehog secreted by Purkinje cells scales the expansion of granule cells and interneurons to produce functional local circuits and the proper folded morphology of the cerebellum.

developmental biology↗

bioRxiv2018

Connectomics of the zebrafish’s lateral-line neuromast reveals wiring and miswiring in a simple microcircuit

The lateral-line neuromast of the zebrafish displays a restricted, consistent pattern of innervation that facilitates the comparison of microcircuits across individuals, developmental stages, and genotypes. We used serial blockface scanning electron microscopy to determine from multiple specimens the neuromast connectome, or comprehensive set of connections between hair cells and afferent and efferent nerve fibers. This analysis delineated a complex but consistent wiring pattern with three striking characteristics: each nerve terminal is highly specific in receiving innervation from hair cells of a single directional sensitivity; the innervation is redundant; and the terminals manifest a hierarchy of dominance. Mutation of the canonical planar-cell-polarity gene vangl2, which decouples the asymmetric phenotypes of sibling hair-cell pairs, results in randomly positioned, randomly oriented sibling cells that nonetheless retain specific wiring. Because larvae that overexpress Notch exhibit uniformly oriented, uniformly innervating hair-cell siblings, wiring specificity is mediated by the Notch signaling pathway.

developmental biology↗

bioRxiv2018

Drosophila mef2 is essential for normal mushroom body and wing development.

MEF2 (myocyte enhancer factor 2) transcription factors are found in the brain and muscle of insects and vertebrates and are essential for the differentiation of multiple cell types. We show that in the fruitfly Drosophila, MEF2 is essential for normal development of wing veins, and for mushroom body formation in the brain. In embryos mutant for D-mef2, there was a striking reduction in the number of mushroom body neurons and their axon bundles were not detectable. D-MEF2 expression coincided with the formation of embryonic mushroom bodies and, in larvae, expression onset was confirmed to be in post-mitotic neurons. With a D-mef2 point mutation that disrupts nuclear localization, we find that D-MEF2 is restricted to a subset of Kenyon cells that project to the /{beta}, and {gamma} axonal lobes of the mushroom bodies, but not to those forming the /{beta} lobes. Our findings that ancestral mef2 is specifically important in dopamine-receptive neurons has broad implications for its function in mammalian neurocircuits.

developmental biology↗

bioRxiv2018

Restriction of chromatin accessibility is necessary for appropriate enhancer expression

Tissue specific gene expression underpins cell type diversity, and arises from the cooperative activities of transcription factors and the chromatin landscape. It has been previously demonstrated that enhancers with specific arrangements of transcription factor binding motifs can bring together commonly and specifically expressed factors in order to stabilize chromatin accessibility and drive spatially restricted reporter expression within different regions of the CNS. However, when reporters were used to analyse the activity of enhancers bound differentially by a common factor in the endoderm and CNS, several examples of non-tissue specific reporter expression were observed. In order to judge whether or not this may have been due to the unregulated chromatin environment of exogenously delivered enhancer reporters, here we have analysed the chromatin landscape of cells from the CNS and endodermal tissues and find that this reflects neighbouring gene expression to a greater degree than transcription factor binding. This work demonstrates that chromatin accessibility plays an essential role in defining enhancer activity in distantly related cell types.

developmental biology↗

bioRxiv2018

Sex Determination in the garden lizard, Calotes versicolor: Is Environment a Factor?

The Indian garden lizard, C. versicolor, is known to lack sex chromosomes (Singh, 1974, Ganesh et al 1997). A report from the tropical southern India (Inamdar et al., 2012), claims a TSD (FMFM) mechanism in this species in which the male/female ratio in embryos oscillates within a range of 3-4{degrees}C. The present study presents results of experiments done in 4 consecutive breeding seasons of C. versicolor belonging to the subtropical/temperate climate of northern region of India. Eggs were grown at different temperatures (at 24.5/28/31.5{degrees}C) or under seminatural conditions. Another set of eggs was exposed to fadrozol, an aromatase inhibitor (AI), or Lithium Chloride (inhibitor of GSK3 enzyme inducing Wnt4-dependent Beta-Catenin). Results confirm our earlier finding that in this subtropical population of C. versicolor, temperature does not regulate gonadal differentiation but AI-induces sex reversal to the male sex (Ganesh and Raman, 1995, Ganesh et al 1999). We also report lack of any effect of Sox9 inhibitor on sexual differentiation which may be due to inadequate quantity or mode of application. Importantly, we report the serendipitous observation that in each year almost all the embryos/hatchlings were of the same sex ( females in 2013, 15, 16 and males in 2014) regardless of the rearing condition and duration of incubation. Obviously, parthenogenesis is not the cause of it. In the absence of an obvious reason to explain this pattern, we surmise that in this north Indian population of C. versicolor, female is the default sex, and certain epigenetic regulators could modulate the sexual differentiation of the individual.

developmental biology↗

bioRxiv2018

MECP2 duplication and mutations impair NSCs differentiation via miR-197 regulated ADAM10

How MECP2 (Methyl-CpG-binding protein 2) duplication affects cortex development remains elusive. We found that elevated MeCP2 expression promotes neurogenesis during cortex development in Tg(MECP2) mouse brain. Ectopic expression of MeCP2 in NPCs inhibits ADAM10 and hence compromises the NOTCH pathway during NPC differentiation. MeCP2 up-regulates miR-197 to down-regulate ADAM10. The enhanced NPC differentiation/migration in Tg(MECP2) embryonic brain can be repressed by overexpression of ADAM10 or a miR-197 inhibitor.\n\nConsistently, the reduced neurogenesis induced by three rare MeCP2 missense mutations (H371R, E394K, G428S) identified in a Han Chinese autism spectrum disorders (ASD) cohort, can be reversed by miR-197 both in vitro and in vivo. Our results revealed that a regulatory axis involving MeCP2, miR-197, ADAM10, and NOTCH signaling is critical for neurogenesis, which is affected by both MeCP2 duplication and mutation.

developmental biology↗

bioRxiv2018

Specific Oxylipins Enhance Vertebrate Hematopoiesis via the Receptor GPR132

Epoxyeicosatrienoic acids (EETs) are endogenous lipid signaling molecules with cardioprotective and vasodilatory actions. We recently showed that exogenous addition of 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via a G-protein coupled receptor(s), and significant research supports the existence of a specific high-affinity receptor. Identification of a hematopoietic specific EET receptor would enable genetic interrogation of the EET signaling pathway and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify the EET receptor based on the expression of GPCRs in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are commonly expressed in three EET-responsive human cell lines, but not expressed in an EET-unresponsive line. Of these candidates, only GPR132 showed EET-responsiveness in vitro using a luminescence-based assay for {beta}-arrestin recruitment. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to the putative high-affinity EET receptor, GPR132 is reported to have affinity for additional fatty acids in vitro, and we found that these same fatty acids enhance hematopoietic stem cell specification in the zebrafish. We conducted structure-activity relationship analyses using both in vitro and in vivo assays on diverse medium chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, while unoxygenated or saturated fatty acids had lower activity. Absence of the carboxylic acid moiety prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of polyunsaturated, oxygenated fatty acids to enhance both embryonic and adult hematopoiesis.

developmental biology↗

bioRxiv2018

The gene regulatory basis of genetic compensation during neural crest induction

BackgroundThe neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors but detailed interactions within the network remain to be resolved.\n\nResultsWe have used gene knockout and RNA sequencing strategies to dissect NC differentiation in zebrafish. We establish that initiation of the NC GRN takes place just after genome activation. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that although a single allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, expression of many target genes remains abnormal and sensitive to tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation.\n\nConclusionsStepwise genetic ablation of the NC identifies the core gene module required for neural crest induction. This work extends and refines the NC GRN while also uncovering the complex transcriptional basis of genetic compensation via paralogues.

developmental biology↗

bioRxiv2018

Distinct temporal requirements for Sonic hedgehog signaling in development of the tuberal hypothalamus

ABSTRACTSonic hedgehog (Shh) plays well characterized roles in the development of several regions of the brain and spinal cord, but its functions in the hypothalamus have been more difficult to elucidate due to the complex neuroanatomy of this brain area. Here, we utilize fate-mapping and conditional deletion models in mice to define requirements for dynamic Shh activity at distinct stages of tuberal hypothalamic development, a brain region with important homeostatic functions. At early time points, Shh signaling regulates dorsoventral patterning, neurogenesis, and the size of the ventral midline. Fate mapping experiments demonstrate that Shh expressing and responsive progenitors contribute to distinct neuronal subtypes, accounting for some of the cellular heterogeneity in tuberal hypothalamic nuclei. Conditional deletion of the Hedgehog transducer Smoothened (Smo), after dorsoventral patterning has been established, reveals that Shh signaling is necessary to maintain proliferation and progenitor identity during peak periods of hypothalamic neurogenesis. We also find that mosaic disruption of Smo causes a non-cell autonomous gain in Shh signaling activity in neighboring wild type cells, suggesting a mechanism for the pathogenesis of hypothalamic hamartomas, a benign tumor that forms during hypothalamic development.\n\nSUMMARY STATEMENTRequirements for dynamic Sonic hedgehog activity at distinct stages of tuberal hypothalamic development are defined using fate-mapping and conditional deletion models in mice.

developmental biology↗

bioRxiv2018

The ubiquitin ligase CRL3Kelch targets HtsRC to organize the Drosophila ring canal cytoskeleton

During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.

developmental biology↗

bioRxiv2018

A neuronal MAP kinase constrains growth of a C. elegans sensory dendrite throughout the life of the organism

Neurons develop elaborate morphologies that provide a model for understanding cellular architecture. By studying C. elegans sensory dendrites, we previously identified genes that act to promote the extension of ciliated sensory dendrites during embryogenesis. Interestingly, the nonciliated dendrite of the oxygen-sensing neuron URX is not affected by these genes, suggesting it develops through a distinct mechanism. Here, we use a visual forward genetic screen to identify mutants that affect URX dendrite morphogenesis. We find that disruption of the MAP kinase MAPK-15 or the {beta}H-spectrin SMA-1 causes a phenotype opposite to what we had seen before: dendrites extend normally during embryogenesis but begin to overgrow as the animals reach adulthood, ultimately extending up to 150% of their normal length. SMA-1 is broadly expressed and acts non-cell-autonomously, while MAPK-15 is expressed in many sensory neurons including URX and acts cell-autonomously. MAPK-15 acts at the time of overgrowth, localizes at the dendrite ending, and requires its kinase activity, suggesting it acts locally in time and space to constrain dendrite growth. Finally, we find that the oxygen-sensing guanylate cyclase GCY-35, which normally localizes at the dendrite ending, is localized throughout the overgrown region, and that overgrowth can be suppressed by overexpressing GCY-35 or by genetically mimicking elevated cGMP signaling. These results suggest that overgrowth may correspond to expansion of a sensory compartment at the dendrite ending, reminiscent of the remodeling of sensory cilia or dendritic spines. Thus, in contrast to established pathways that promote dendrite growth during early development, our results reveal a distinct mechanism that constrains dendrite growth throughout the life of the animal, possibly by controlling the size of a sensory compartment at the dendrite ending.\n\nAUTHOR SUMMARYLewis Carrolls Alice told the Caterpillar, \"Being so many different sizes in a day is very confusing.\" Like Alice, the cells of our bodies face a problem in size control - they must become the right size and remain that way throughout the life of the organism. This problem is especially relevant for nerve cells (neurons), as the lengths of their elaborate dendrites determine the connections they can make. To learn how neurons control their size, we turned not to a Caterpillar but to a worm: the microscopic nematode C. elegans, in which single neurons can be easily visualized and the length of each dendrite is highly predictable across individuals. We focused on a single dendrite, that of the oxygen-sensing neuron URX, and we identified two genes that control its length. When these genes are disrupted, the dendrite develops correctly in embryos but then, like Alice, grows too much, eventually extending up to 1.5x its normal length. Thus, in contrast to known pathways that promote the initial growth of a dendrite early in development, our results help to explain how a neuron maintains its dendrite at a consistent length throughout the animals life.

developmental biology↗

bioRxiv2018

N-linked glycosylation of the antagonist Short gastrulation increases the functional complexity of BMP signals

Disorders of N-linked glycosylation are increasingly reported in the literature. However, targets responsible for the associated developmental and physiological defects are largely unknown. Bone Morphogenetic Proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. Here we investigate the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). We identify conserved N-glycosylated sites and describe the effect of mutating these residues on BMP pathway activity in Drosophila. Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, we provide evidence that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.\n\nSummary StatementN-glycosylation restricts the function of Short gastrulation during Drosophila development by controlling the amount of extracellular protein. This adds another layer of complexity to regulation of Bone Morphogenetic Protein signals.

developmental biology↗

bioRxiv2018

HIPPO signaling provides a fail-safe for resolving embryonic cell fate conflicts during establishment of pluripotency in vivo

During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway segregates pluripotent and extraembryonic fates by controlling cell positioning as well as expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.

developmental biology↗

bioRxiv2018

Frequency and synchrony of actomyosin oscillation during PCP-dependent convergent extension

Oscillatory actomyosin flows play a key role in single cell migration and in collective cell movements that shape invertebrates embryos, but the role of such oscillations in vertebrate morphogenesis remains poorly defined. Here, data from mathematical modeling and in vivo 4D imaging of actomyosin in the Xenopus gastrula suggest that oscillatory actomyosin contractions are a general feature of convergent extension by junction shrinking. We show that synchronous intracellular flows link two spatially distinct populations of actomyosin within individual cells, but that oscillations are asynchronous between neighboring cells that share a shrinking cell-cell junction. We also show that the core PCP protein Prickle2 displays a parallel oscillatory behavior and is required for tuning the frequency of actomyosin contractions, indicating that PCP signaling controls not only the orientation of actomyosin contractions, but also their frequency. Together, these data provide new insights into the function and control of oscillatory actomyosin contractions in collective cell movement.

developmental biology↗

bioRxiv2018

On the Development of Sesamoid Bones

Sesamoid bones are a special group of small auxiliary bones that form in proximity to joints and contribute to their stability and function. Sesamoid bones display high degree of variability in size, location, penetrance and anatomical connection to the main skeleton across vertebrate species. Therefore, providing a comprehensive developmental model or classification system for sesamoid bones is challenging. Here, we examine the developmental mechanisms of three anatomically different sesamoid bones, namely patella, lateral fabella and digit sesamoids. Through a comprehensive comparative analysis at the cellular, molecular and mechanical levels, we demonstrate that all three types of sesamoid bones originated from Sox9+/Scx+ progenitors under the regulation of TGF{beta} and independent of mechanical stimuli from muscles. We show that BMP4 was necessary specifically for differentiation of patella but not of lateral fabella or digit sesamoids, whereas BMP2 regulated the growth of all examined sesamoids. Next, we show that whereas patella and digit sesamoids initially formed in juxtaposition to long bones, the lateral fabella formed independently at a distance. Finally, we provide evidence suggesting that while patella detached from the femur by formation of a synovial joint, digit sesamoids detached from the phalanx by a fibrocartilage joint. Collectively, these findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, thereby advancing our understanding of their evolutionary plasticity.

developmental biology↗

bioRxiv2018

Comparison of the 3-D patterns of the parasympathetic nervous system in the lung at late developmental stages between mouse and chicken

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds.\n\nHighlightsO_LI3-D patterns of parasympathetic nerves are visualized in mouse and chicken lungs.\nC_LI\n\nComparison of these patterns reveals three prominent similarities between mouse and chicken: O_LIVAChT-positive postganglionic fibers and ganglia are widely distributed in the lung.\nC_LIO_LIGas exchange units are devoid of parasympathetic nerves.\nC_LIO_LIParasympathetic nerves are in close association with smooth muscle cells.\nC_LI

developmental biology↗

bioRxiv2018

The phylogenetically distinct early human embryo

Is the human early embryo unique in lacking an inner cell-mass (ICM) and having parallel development? We reanalyse single-cell transcriptomic data and stain human embryos in situ to reveal both classical step-wise development and a transcriptomically homologous ICM. This apparent classicism obscures phylogenetic singularity: unlike mice, human epiblast has self-renewal hallmarks and we have abundant blastocyst non-committed cells (NCCs), part of an apoptosis-mediated purging process. The transcriptomes of the pluripotent cells are fast evolving, in large part owing to endogenous retrovirus H (ERVH) activity, rendering all primate embryos unique. Each species is characterised by the ERVHs that are active and the neighbour genes whose expression are modulated. ERVH is associated with recent major gene expression gain and loss events of pluripotency{-}associated genes. Not least through lack of HERVH expression, the current portfolio of naive cultures, putative in vitro mimics of pluripotent cells, are both developmentally and phylogenetically "confused". O_LIAnalysis of single cell transcriptomics and in situ stainings uncover, and enable characterization of, human inner cell mass (ICM) C_LIO_LICell purging via apoptosis defines a phylogenetically restricted class of blastocyst non-committed cells (NCCs), whereas HERVs in conjunction with host defence mark the committed cells of ICM C_LIO_LIFast transcriptome evolution is particular to the pluripotent epiblast and is mostly due to the primate-specific transposable element, HERVH C_LIO_LICurrent naive cultures dont reflect human uniqueness being phylogenetically and developmentally "confused". C_LI

developmental biology↗