SEARCH · bioRxiv Science

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,423 records · Page 79Linked to original sources

bioRxiv2019

Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

Small RhoGTPases and Myosin-II direct cell shape changes and movements during tissue morphogenesis. Their activities are tightly regulated in space and time to specify the desired pattern of contractility that supports tissue morphogenesis. This is expected to stem from polarized surface stimuli and from polarized signaling processing inside cells. We examined this general problem in the context of cell intercalation that drives extension of the Drosophila ectoderm. In the ectoderm, G protein coupled receptors (GPCRs) and their downstream heterotrimeric G proteins (G and G{beta}{gamma}) activate Rho1 both medial-apically, where it exhibits pulsed dynamics, and at junctions, where its activity is planar polarized (Kerridge et al., 2016; Munjal et al., 2015). However, the mechanisms responsible for polarizing Rho1 activity are unclear. In particular, it is unknown how Rho1 activity is controlled at junctions. We report a division of labor in the mechanisms of Rho1 activation in that distinct guanine exchange factors (GEFs), that serve as activators of Rho1, operate in these distinct cellular compartments. RhoGEF2 acts uniquely to activate medial-apical Rho1. Although RhoGEF2 is recruited both medial-apically and at junctions by G12/13-GTP, also called Concertina (Cta) in Drosophila, its activity is restricted to the medial-apical compartment. Furthermore, we characterize a novel RhoGEF, p114RhoGEF/Wireless (Wrl), and report its requirement for cell intercalation in the extending ectoderm. p114RhoGEF/Wireless activates Rho1 specifically at junctions. Strikingly it is restricted to adherens junctions and is under G{beta}13F/G{gamma}1 control. G{beta}13F/G{gamma}1 activates junctional Rho1 and exerts quantitative control over planar polarization of Rho1. In particular, overexpression of G{beta}13F/G{gamma}1 leads to hyper planar polarization of Rho1 and MyoII. Finally, we found that p114RhoGEF/Wireless is absent in the mesoderm, arguing for a tissue-specific control over junctional Rho1 activity. These results shed light on the mechanisms of polarization of Rho1 activity in different cellular compartments and reveal that distinct GEFs are sensitive tuning parameters of cell contractility in remodeling epithelia.

developmental biology↗

bioRxiv2019

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Therapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-target events. While recent work in rodents outlines favorable conditions for specific editing and uses a trio design to control for the contribution of natural genome variation, the potential for CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we performed whole exome sequencing (WES) on two generations of offspring derived from the same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation (F1). We did not observe an increase in the number of transmissible variants in edited individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not precipitate an inflation of deleterious point mutations.

developmental biology↗

bioRxiv2019

In the chick embryo, estrogen can induce chromosomally male ZZ left gonad epithelial cells to form an ovarian cortex which supports oogenesis

In chickens, as in most amniotes, the first morphogenetic event in ovary differentiation is the formation of two distinct domains: a steroidogenic core, the medulla, overlain by the germ cell niche, the cortex. This process normally starts immediately after sex determination in the ZW embryos, substantially before the progression of germ cells into meiosis. In order to address the extent to which the cortical domain depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering estrogen levels of ZZ and ZW embryos in ovo. We found that both chomosomally female and male cells can be induced to form the cortical domain and that this can form relatively independently of the phenotypic sex of the medulla as long as estrogen is provided. We also show that the cortex promoting activity of estrogen signalling is mediated via Estrogen Receptor alpha within the left gonad epithelium. Therefore, either a ZW or ZZ cortical domain can provide an adequate niche to allow the germ cells to progress into meiosis. However, the presence of a medulla with an \"intersex\" or male phenotype may compromise this progression, causing cortical germ cells to remain in an immature state in the embryo.

developmental biology↗

bioRxiv2019

DUX is a non-essential synchronizer of zygotic genome activation

Some of the earliest transcripts produced in fertilized human and mouse oocytes code for DUX, a double homeodomain protein that promotes embryonic genome activation (EGA). Deleting Dux by genome editing at the 1- to 2-cell stage in the mouse impairs EGA and blastocyst maturation. Here, we demonstrate that mice carrying homozygous Dux deletions display markedly reduced expression of DUX target genes and defects in both pre- and post-implantation development, with notably a disruption of the pace of the first few cell divisions and significant rates of late embryonic mortality. However, some Dux-/- embryos give raise to viable pups, indicating that DUX is important but not strictly essential for embryogenesis.\n\nSummary statementMurine DUX regulates transcription in the first embryonic cell divisions but its not necessary for embryogenesis

developmental biology↗

bioRxiv2019

GCNA interacts with Spartan and Topoisomerase II to regulate genome stability.

GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan/DVC-1 and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. We show that GCNA and Topoisomerase 2 (Top2) physically interact and colocalize on condensed chromosomes during mitosis, when Spartan is not present. We show that C. elegans gcna-1 mutants are sensitive to Top2 poison and accumulate mutations consistent with low fidelity repair of DNA damage, leading to loss of fitness and fertility over generations. We also demonstrate that mouse GCNA interacts with TOP2, and Gcna-mutant mice exhibit abnormalities consistent with the inability to process DPCs, including chromatin condensation and crossover defects. Together, our findings provide evidence that GCNA maintains genomic integrity by processing Top2 DPCs in the germline and early embryo, where the genome is challenged with an increased DPC burden.

developmental biology↗

bioRxiv2019

Interkinetic nuclear migration in the zebrafish retina as a diffusive process

A major hallmark of neural development is the oscillatory movement of nuclei between the apical and basal surfaces of the neuroepithelium during the process of interkinetic nuclear migration (IKNM). Here, we employ long-term, rapid lightsheet and two-photon imaging of Zebrafish retinas in vivo during early development to uncover the physical processes that govern the behavior of nuclei during IKNM. These images allow the capture of reliable tracks of nuclear movements and division during early retinogenesis for many tightly packed nuclei. These tracks are then used to create and test a theory of retinal IKNM as a diffusive process across a nuclear concentration gradient generated by the addition of new nuclei at the apical surface. The analysis reveals the role of nuclear packing at the apical surface on the migration dynamics of nuclei, provides a robust quantitative explanation for the distribution of nuclei across the retina, and may have implications for stochastic fate choice in this system.

developmental biology↗

bioRxiv2019

GCNA preserves genome integrity and fertility across species

The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While these cells employ a number of known repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ Cell Nuclear Acidic Peptidase (GCNA) as a highly conserved regulator of genome stability in flies, worms, zebrafish, and humans. GCNA contains a long acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, GCNA mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, most of GCNAs function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.\n\nHighlightsGCNA ensures genomic stability in germ cells and early embryos across species\n\nGCNA limits replication stress and DNA double stranded breaks\n\nGCNA restricts DNA-Protein Crosslinks within germ cells and early embryos\n\nThe IDR and SprT domains of GCNA govern distinct aspects of genome integrity\n\nGraphic Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC=\"FIGDIR/small/570804_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@1928d82org.highwire.dtl.DTLVardef@885ff2org.highwire.dtl.DTLVardef@15322c9org.highwire.dtl.DTLVardef@110c615_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2019

The protein repertoire in early vertebrate embryogenesis

We present an unprecedentedly comprehensive characterization of protein dynamics across early development in Xenopus laevis, available immediately via a convenient Web portal. This resource allows interrogation of the protein expression data in conjunction with other data modalities such as genome wide mRNA expression. This study provides detailed data for absolute levels of [~]14K unique Xenopus proteins representing homologues of [~]9K unique human genes - a rich resource for developmental biologists. The purpose of this manuscript is limited to presenting and releasing the data browser.\n\nHighlightsO_LIRelative protein expression from stage IV oocyte, blastula, gastrula, neurula, and early organogenesis\nC_LIO_LIBiological triplicates with confidence intervals on protein expression reflect certainty in dynamic patterns\nC_LIO_LIConvenient time-series Web-browser integrated with the multi-media Xenbase portal\nC_LIO_LIGene-symbol search and multi-gene protein/mRNA juxtaposition capabilities\nC_LI

developmental biology↗

bioRxiv2019

Fenestrae-associated protein Plvap regulates the rate of blood-borne proteins passage into the hypophysis

To maintain body homeostasis, endocrine systems must detect and integrate a multitude of blood-borne peripheral signals. This is mediated by specialized permeable pores in the endothelial membrane, dubbed fenestrae. Plasmalemma vesicles-associated protein (Plvap) is located in the fenestral diaphragm and is thought play a role in the selective passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. Here, we studied the development of fenestrated capillaries in a major neuroendocrine interface between the blood and brain, namely the hypophysis. Using a transgenic permeability biosensor to visualize the vascular excretion of a genetically tagged plasma protein (DBP-EGFP), we show that the developmental acquisition of vascular permeability is associated with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis of the hypophyseal vasculature revealed that plvapb mutants display deficiencies in fenestral and stomatal diaphragms as well as increased density of fenestrae, but not of caveolae. Measurements of DBP-EGFP dynamics in live plvapb mutant larvae provided a direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. Overall, we present the regulatory role of Plvap in the development of blood-borne protein detection machinery in a major neuroendocrine interface between the brain and the general circulation.

developmental biology↗

bioRxiv2019

An atlas of anterior hox gene expression in the embryonic sea lamprey head: hox-code evolution in vertebrates

In the hindbrain and the adjacent cranial neural crest (NC) cells of jawed vertebrates (gnathostomes), nested and segmentally-restricted domains of Hox gene expression provide a combinatorial Hox-code for specifying regional properties during head development. Extant jawless vertebrates, such as the sea lamprey (Petromyzon marinus), can provide insights into the evolution and diversification of this Hox-code in vertebrates. There is evidence for gnathostome-like spatial patterns of Hox expression in lamprey; however, the expression domains of the majority of lamprey hox genes from paralogy groups (PG) 1-4 are yet to be characterized, so it is unknown whether they are coupled to hindbrain segments (rhombomeres) and NC. In this study, we systematically describe the spatiotemporal expression of all 14 sea lamprey hox genes from PG1-PG4 in the developing hindbrain and pharynx to investigate the extent to which their expression conforms to the archetypal gnathostome hindbrain and pharyngeal hox-codes. We find many similarities in Hox expression between lamprey and gnathostome species, particularly in rhombomeric domains during hindbrain segmentation and in the cranial neural crest, enabling inference of aspects of Hox expression in the ancestral vertebrate embryonic head. These data are consistent with the idea that a Hox regulatory network underlying hindbrain segmentation is a pan vertebrate trait. We also reveal differences in hindbrain domains at later stages, as well as expression in the endostyle and in pharyngeal arch (PA) 1 mesoderm. Our analysis suggests that many Hox expression domains that are observed in extant gnathostomes were present in ancestral vertebrates but have been partitioned differently across Hox clusters in gnathostome and cyclostome lineages after duplication.

developmental biology↗

bioRxiv2019

The Developmental Transcriptome for Lytechinus variegatus Exhibits Temporally Punctuated Gene Expression Changes

Embryonic development is arguably the most complex process an organism undergoes during its lifetime, and understanding this complexity is best approached with a systems-level perspective. The sea urchin has become a highly valuable model organism for understanding developmental specification, morphogenesis, and evolution. As a non-chordate deuterostome, the sea urchin occupies an important evolutionary niche between protostomes and vertebrates. Lytechinus variegatus (Lv) is an Atlantic species that has been well studied, and which has provided important insights into signal transduction, patterning, and morphogenetic changes during embryonic and larval development. The Pacific species, Strongylocentrotus purpuratus (Sp), is another well-studied sea urchin, particularly for gene regulatory networks (GRNs) and cis-regulatory analyses. A well-annotated genome and transcriptome for Sp are available, but similar resources have not been developed for Lv. Here, we provide an analysis of the Lv transcriptome at 11 timepoints during embryonic and larval development. The data indicate that the gene regulatory networks that underlie specification are well-conserved among sea urchin species. We show that the major transitions in variation of embryonic transcription divide the developmental time series into four distinct, temporally sequential phases. Our work shows that sea urchin development occurs via sequential intervals of relatively stable gene expression states that are punctuated by abrupt transitions.

developmental biology↗

bioRxiv2019

Constrained Transcriptional Polarity In The Organization Of Mammalian Hox Gene Clusters

In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters towards a tighter structure during the evolution of the vertebrate lineage. Here we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster such as to invert one or several transcription unit(s), with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.

developmental biology↗

bioRxiv2019

PRDM16 establishes lineage-specific transcriptional program to promote temporal progression of neural progenitors in the mouse neocortex

Radial glia (RG) in the neocortex sequentially generate distinct subtypes of projection neurons, accounting for the diversity and complex assembly of cortical neural circuits. Mechanisms that drive the rapid and precise temporal progression of RG are beginning to be elucidated. Here we reveal that the RG-specific transcriptional regulator PRDM16 promotes the transition of early to late phases of neurogenesis in the mouse neocortex. Prdm16 mutant RG delays the timely progression of RG, leading to defective cortical laminar organization. We show that PRDM16 regulates expression of neuronal specification genes and a subset of genes that are dynamically expressed between mid-and late-neurogenesis. Our genomic analysis suggests that PRDM16 suppresses target gene expression through maintaining chromatin accessibility of permissive enhancers. Altogether, our results demonstrate a critical role of PRDM16 in establishing stage-specific gene expression program of RG during cortical neurogenesis. These findings also support a model where progenitor cells are primed with daughter cell gene expression program for rapid cell differentiation.

developmental biology↗

bioRxiv2019

A pivotal genetic program controlled by thyroid hormone during the maturation of GABAergic neurons in mice

In mammals, brain development is critically dependent on proper thyroid hormone signaling, via the TR1 nuclear receptor. However, the downstream mechanisms by which TR1 impacts brain development are currently unknown, notably because this receptor is expressed ubiquitously from early stages of development. In order to better define the function of TR1 in the developing brain, we used mouse genetics to induce the expression of a dominant-negative mutation of the receptor specifically in GABAergic neurons, the main inhibitory neurons in the brain, which were previously identified as sensitive to hypothyroidism. This triggered post-natal epileptic seizures, reflecting a profound impairment of GABAergic neuron maturation in different brain areas. Analysis of transcriptome and TR1 cistrome also allowed us to identify a small set of genes, the transcription of which is upregulated by TR1 in GABAergic neurons during post-natal maturation of the striatum and which probably play an important role during neurodevelopment. Thus, our results point to GABAergic neurons as direct targets of thyroid hormone during brain development and suggest that many defects seen in hypothyroid brains may be secondary to GABAergic neuron malfunction.

developmental biology↗

bioRxiv2019

Satb2 regulates proliferation and nuclear integrity of pre-osteoblasts

Special AT-rich sequence binding protein 2 (Satb2) is a matrix attachment region (MAR) binding protein. Satb2 impacts skeletal development by regulating gene transcription required for osteogenic differentiation. Although its role as a high-order transcription factor is well supported, other roles for Satb2 in skeletal development remain unclear. In particular, the impact of dosage sensitivity (heterozygous mutations) and variance on phenotypic severity is still not well understood. To further investigate molecular and cellular mechanisms of Satb2-mediated skeletal defects, we used the CRISPR/Cas9 system to generate Satb2 mutations in MC3T3-E1 cells. Our data suggest that, in addition to its role in differentiation, Satb2 regulates progenitor proliferation. We also find that mutations in Satb2 cause chromatin defects including nuclear blebbing and donut-shaped nuclei. These defects may contribute to a slight increase in apoptosis in mutant cells, but apoptosis is insufficient to explain the proliferation defects. Satb2 expression exhibits population-level variation and is mostly highly expressed from late G1 to late G2. Based on these data, we hypothesize that Satb2 may regulate proliferation through two separate mechanisms. First, Satb2 may regulate the expression of genes necessary for cell cycle progression in pre-osteoblasts. Second, similar to other MAR-binding proteins, Satb2 may participate in DNA replication. Deficiencies in either of these processes could reduce the pace of cell cycle progression and contribute to nuclear damage. We also hypothesize that Satb2-mediated proliferation defects may be buffered in some genetic backgrounds, which provides some explanation for differences in severity of skeletal defects. Further elucidation of the role of Satb2 in proliferation has potential impacts on our understanding of both skeletal defects and cancer.

developmental biology↗

bioRxiv2019

Multi-enhancer transcriptional hubs confer phenotypic robustness

We had previously shown in Drosophila melanogaster embryos that low-affinity Ultrabithorax (Ubx)-responsive shavenbaby (svb) enhancers drive robust expression using localized transcriptional environments and that active svb enhancers tended to colocalize, even when placed on different chromosomes (Tsai et al., 2017). Here, we test the hypothesis that these multi-enhancer \"hubs\" improve robustness by increasing transcription factor retention near transcription sites. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, we observed lower Ubx concentration and transcriptional output in this deletion allele. Transcription sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer against environmental stresses and genetic perturbations, providing a mechanism for phenotypical robustness.\n\nImpact statementMultiple enhancers in physical proximity can reinforce shared transcriptional \"hubs\" to retain transcription factors, providing a buffer during environmental stresses and genetic perturbations to preserve phenotypic robustness.

developmental biology↗

bioRxiv2019

Lumen expansion facilitates epiblast-primitive endoderm fate specification in the mouse blastocyst formation.

Mouse blastocyst formation involves lumen formation and cell fate specification. While many studies have investigated how the blastocyst cell lineages are specified through genetics and signaling, studies into the potential role of the fluid lumen have yet to be conducted. We discover that blastocyst fluid emerges by secretion of cytoplasmic vesicles to intercellular space in addition to trans-epithelial flow. We observe that the beginning of epiblast and primitive endoderm spatial segregation directly follows lumen coalescence. Notably, we show that perturbing lumen expansion by pharmacological and biophysical means impair the specification and spatial segregation of primitive endoderm cells within the blastocyst. Combined, our results suggest that blastocyst lumen expansion plays a critical role in guiding cell fate specification and positioning. As epithelial tissues typically form lumina, lumen expansion may provide a general mechanism of cell fate control in many tissues.

developmental biology↗

bioRxiv2019

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a key phenotype observed in DiGeorge syndrome patients and are caused by a microdeletion on chromosome 22q11. Heterozygosity of TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key component for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9 deficient mice are born with complex cardiovascular malformations affecting the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared to Tbx1 heterozygous mice. Using a novel Pax9Cre allele we demonstrated that the site of this Tbx1-Pax9 genetic interaction is in the pharyngeal endoderm, therefore revealing that a Tbx1/Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for critical tissue interactions during normal morphogenesis of the pharyngeal arch artery system.\n\nSummary statementPax9 is required for outflow tract and aortic arch development, and functions together with Tbx1 in the pharyngeal endoderm for 4th arch artery formation.

developmental biology↗