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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,117 records · Page 62Linked to original sources

bioRxiv2018

Glass confers rhabdomeric photoreceptor identity in Drosophila, but not across all metazoans

Across metazoans, visual systems employ different types of photoreceptor neurons to detect light. These include rhabdomeric PRs, which exist in distantly related phyla and possess an evolutionarily conserved phototransduction cascade. While the development of rhabdomeric PRs has been thoroughly studied in the fruit fly Drosophila melanogaster, we still know very little about how they form in other species. To investigate this question, we tested whether the transcription factor Glass, which is crucial for instructing rhabdomeric PR formation in Drosophila, may play a similar role in other metazoans. Glass homologues exist throughout the animal kingdom, indicating that this protein evolved prior to the metazoan radiation. Interestingly, our work indicates that glass is not expressed in rhabdomeric photoreceptors in the planarian Schmidtea mediterranea nor in the annelid Platynereis dumerilii. Combined with a comparative analysis of the Glass DNA-binding domain, our data suggest that the fate of rhabdomeric PRs is controlled by Glass-dependent and Glass-independent mechanisms in different animal clades.

developmental biology↗

bioRxiv2018

A small proportion of Talin molecules transmit forces to achieve muscle attachment in vivo

Cells in a developing organism are subjected to particular mechanical forces, which shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is thus an important question, which has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Hence, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing three FRET-based Talin tension sensors reporting different force levels between 1 and 11 pN enabled us to quantify physiologically-relevant, molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension increases. Concomitantly, Talin concentration at attachment sites increases five-fold as quantified by fluorescence correlation spectroscopy, suggesting that only few Talin molecules are mechanically engaged at any given time. We therefore propose that high tissue forces are shared amongst a large excess of adhesion molecules of which less than 15% are experiencing detectable forces at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.

developmental biology↗

bioRxiv2018

Characteristic Localization of Neuronatin in Rat Tissues

Neuronatin (Nnat) is expressed in the pituitary, pancreas, and other tissues; however, the function of NNAT is still unclear. Recent studies have demonstrated that NNAT is localized in the sex determining region Y-box 2-positive stem/progenitor cells in the developing rat pituitary primordium and is downregulated during differentiation into mature hormone-producing cells. Moreover, NNAT is widely localized in subcellular organelles, excluding the Golgi. Here, we further evaluated NNAT expression and intracellular localization in embryonic and postnatal rat tissues such as the pancreas, tongue, whisker hair follicle, and testis. Immunohistochemistry showed that NNAT was localized in undifferentiated cells (i.e., epithelial basal cells and basement cells in the papillae of the tongue and round and elongated spermatids of the testis) as well as in differentiated cells (insulin-positive cells and exocrine cells of the pancreas, taste receptor cells of the fungiform papilla, the inner root sheath of whisker hair follicles, and spermatozoa). Additionally, NNAT showed novel intracellular localization in acrosomes in the spermatozoa. Because the endoplasmic reticulum (ER) is excluded from spermatozoa and sarco/ER Ca2+-ATPase isoform 2 (SERCA2) is absent from the inner root sheath, these findings suggested that NNAT localization in the ER and its interaction with SERCA2 were cell-or tissue-specific properties.

developmental biology↗

bioRxiv2018

DPPA2 and DPPA4 are necessary to establish a totipotent state in mouse embryonic stem cells

After fertilization of the transcriptionally silent oocyte, expression from both parental chromosomes is launched through so-called zygotic genome activation (ZGA), occurring in the mouse at the 2-cell stage. Amongst the first elements to be transcribed are the Dux gene, the product of which secondarily induces a wide array of ZGA genes, and a subset of evolutionary recent LINE-1 retrotransposons, which regulate chromatin accessibility in the early embryo. The maternally-inherited factors that activate Dux and LINE-1 transcription have so far remained unknown. Here we identify the paralog proteins DPPA2 and DPPA4 as responsible for this process.

developmental biology↗

bioRxiv2018

Competition for space induces cell elimination through compaction-driven ERK downregulation

The plasticity of developing tissues relies on the adjustment of cell survival and growth rate to environmental cues. This includes the effect of mechanical cues on cell survival. Accordingly, compaction of an epithelium can lead to cell extrusion and cell death. This process was proposed to contribute to tissue homeostasis but also to facilitate the expansion of pretumoral cells through the compaction and elimination of the neighbouring healthy cells. However we know very little about the pathways than can trigger apoptosis upon tissue deformation and the contribution of compaction driven death to clone expansion was never assessed in vivo. Using the Drosophila pupal notum and a new live sensor of ERK, we show that tissue compaction induces cell elimination through the downregulation of EGFR/ERK pathway and the upregulation of the pro-apoptotic protein Hid. Those results suggest that the sensitivity of EGFR/ERK pathway to mechanics could play a more general role in the fine tuning of cell elimination during morphogenesis and tissue homeostasis. Secondly, we assessed in vivo the contribution of compaction driven death to pretumoral cell expansion. We found that the activation of the oncogene Ras in clones can also downregulate ERK and activate apoptosis in the neighbouring cells through their compaction, which contributes to Ras clone expansion. The mechanical modulation of EGFR/ERK during growth-mediated competition for space may contribute to tumour progression.

developmental biology↗

bioRxiv2018

GFP-Forked, a genetic reporter for studying Drosophila oocyte polarity

The polarized organization of the Drosophila oocyte can be visualized by examining the asymmetric localization of mRNAs, which is supported by networks of polarized microtubules (MTs). In this study, we used the gene forked, the putative Drosophila homologue of espin, to develop a unique genetic reporter for asymmetric oocyte organization. We generated a null allele of the forked gene using the CRISPRCas9 system and found that forked is not required for determining the axes of the Drosophila embryo. However, ectopic expression of a truncated form of GFP-Forked generated a distinct network of asymmetric Forked, which first accumulated at the oocyte posterior and was then restricted to the anterolateral region of the oocyte cortex in mid-oogenesis. This localization pattern resembled that reported for the polarized MTs network. Indeed, pharmacological and genetic manipulation of the polarized organization of the oocyte showed that the filamentous Forked network diffused throughout the entire cortical surface of the oocyte, as would be expected upon perturbation of oocyte polarization. Finally, we demonstrated that Forked associated with Short-stop and Patronin foci, which assemble non-centrosomal microtubule-organizing centers. Our results thus show that clear visualization of asymmetric GFP-Forked network localization can be used as a novel tool for studying oocyte polarity.\n\nSummary statementThe novel asymmetric Forked network could be used as a genetic reporter for visualizing and studying oocyte polarity.

developmental biology↗

bioRxiv2018

Temporal correlation of elevated PRMT1 gene expression with mushroom body neurogenesis during bumblebee brain development

Proper neural development in insects depends on the controlled proliferation and differentiation of neural precursors. In holometabolous insects, these processes must be coordinated during larval and pupal development. Recently, protein arginine methylation has come into focus as an important mechanism of controlling neural stem cell proliferation and differentiation in mammals. Whether a similar mechanism is at work in insects is unknown. We investigated this possibility by determining the expression pattern of three protein arginine methyltransferase mRNAs (PRMT1, 4 and 5) in the developing brain of bumblebees by in situ hybridisation. We detected expression in neural precursors and neurons in functionally important brain areas throughout development. We found markedly higher expression of PRMT1, but not PRMT4 and PRMT5, in regions of mushroom bodies containing dividing cells during pupal stages at the time of active neurogenesis within this brain area. At later stages of development, PRMT1 expression levels were found to be uniform and did not correlate with actively dividing cells. Our study suggests a role for PRMT1 in regulating neural precursor divisions in the mushroom bodies of bumblebees during the period of neurogenesis.

developmental biology↗

bioRxiv2018

Epiblast formation by Tead-Yap-dependent expression of pluripotency factors and competitive elimination of unspecified cells

The epiblast is a pluripotent cell population first formed in preimplantation embryos and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor Tead and its coactivator Yap regulate expression of pluripotency factors. After specification of the inner cell mass, Yap accumulates in the nuclei and activates Tead. Tead activity is required for strong expression of pluripotency factors and is variable in the forming epiblast. Cells showing low Tead activity are eliminated from the epiblast through cell competition. Pluripotency factor expression and Myc control cell competition downstream of Tead activity. Cell competition eliminates unspecified cells and is required for proper organization of the epiblast. These results suggest that induction of pluripotency factors by Tead activity and elimination of unspecified cells via cell competition ensure the production of an epiblast with naive pluripotency.

developmental biology↗

bioRxiv2018

Hox gene expression in postmetamorphic juveniles of the brachiopod Terebratalia transversa

BackgroundHox genes encode a family of homeodomain containing transcription factors that are clustered together on chromosomes of many Bilateria. Some bilaterian lineages express these genes during embryogenesis in spatial and/or temporal order according to their arrangement in the cluster, a phenomenon referred to as collinearity. Expression of Hox genes is well studied during embryonic and larval development of numerous species; however, relatively few studies focus on the comparison of pre- and postmetamorphic expression of Hox genes in animals with biphasic life cycle. Recently, the expression of Hox genes was described for embryos and larvae of Terebratalia transversa, a rhynchonelliformean brachiopod, which possesses distinct metamorphosis from planktonic larvae to sessile juvenile. During premetamorphic development, T. transversa does not exhibit spatial collinearity and several of its Hox genes are recruited for the morphogenesis of novel structures. In our study we determined expression of Hox genes in post-metamorphic juveniles of T. transversa in order to examine metamorphosis-related changes of expression patterns and to test if Hox genes are expressed in the spatially collinear way in the postmetamorphic juveniles.\n\nResultsHox genes are expressed in a spatially non-collinear manner in juveniles, generally showing similar patterns as ones observed in competent larvae: genes labial and Post1 are expressed in chaetae-related structures, sex comb reduced in the shell forming epithelium, whereas Lox5 and Lox4 in dorso-posterior epidermis. After metamorphosis expression of genes proboscipedia, Hox3, Deformed and Antennapedia becomes restricted to, respectively, shell musculature, prospective hinge rudiments, and pedicle musculature and epidermis.\n\nConclusionsAll developmental stages of T. transversa, including postmetamorphic juveniles, exhibit a spatial non-collinear Hox genes expression with only minor changes observed between pre- and postmetamorphic stages. Our results are concordant with morphological observation that metamorphosis in rhynchonelliformean brachiopods, despite being rapid, is rather gradual. The most drastic changes in Hox genes expression patterns observed during metamorphosis could be explained by the inversion of the mantle lobe, which relocates some of the more posterior larval structures into the anterior edge of the juveniles. Cooption of Hox genes for the morphogenesis of novel structures is even more pronounced in post-metamorphic brachiopods when compared to larvae.

developmental biology↗

bioRxiv2018

A Cyclin A - Myb-MuvB - Aurora B network regulates the choice between mitotic cycles and polyploid endoreplication cycles.

Cells switch to polyploid endoreplication cycles during development, wound healing, and cancer. We used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between developmental and induced endoreplicating cells (devECs and iECs). We found that while only devECs had a dampened E2F1 transcriptome, repression of a Cyclin A - Myb-MuvB - Aurora B mitotic network promoted endoreplication in both devECs and iECs. Cyclin A associated with and activated Myb-MuvB to induce transcription of mitotic genes, with expression of one, Aurora B, being key for mitotic commitment. Knockdown of Cyclin A, Myb, Aurora B, or downstream cytokinetic proteins induced distinct types of endoreplication, suggesting that repression of different mitotic network steps may explain the known diversity of polyploid cycles. These findings reveal how remodeling of a mitotic network promotes polyploid cycles that contribute to development, wound healing, and cancer.

developmental biology↗

bioRxiv2018

Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naive genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/tri methylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo the heterochromatin lacks these modifications which only appear later when rapid embryonic cell cycles slow down at the Mid-Blastula Transition or MBT. Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live-imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase, but then dissociates in mitosis and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation.

developmental biology↗

bioRxiv2018

Drosophila scribble mutant tumors undergo a transition from a growth arrest state to a proliferative state over time

The Drosophila neoplastic tumor suppressor gene (nTSG) mutant tumors have successfully modeled many aspects of human tumor progression. However, the fly nTSG mutant tumors progress rapidly over days. This is in contrast with most human tumors which develop slowly, harbor heterogeneous cell populations for selection and undergo an evolution-like process. Whether the fast-growing fly nTSG mutant tumors have capacity for evolution remains unclear. Through quantitative analysis of the scrib mutant tumor growth, we found that the scrib mutant tumors evolve to display different growth rates and cell cycle profiles over time. Multiple growth-regulatory signaling pathways show quantitative differences in early versus late scrib mutant tumors. These data suggest that the scrib mutant tumors undergo a transition from a growth arrest state to a proliferative state. Through longitudinal single cell RNA (scRNA) data analysis we found that the scrib mutant tumors harbor heterogeneous cell populations likely of distinct proliferative states, which are available for potential selection. This study raises the possibility of studying tumor evolution in a genetically accessible and fast-growing invertebrate tumor model.

developmental biology↗

bioRxiv2018

Re-purposing Ac/Ds transgenic system for CRISPR/dCas9 modulation of enhancers and non-coding RNAs in zebrafish

Due to its genetic amenability coupled with recent advances in genome editing, the zebrafish serves as an excellent model to examine the function of both coding and non-coding elements. Recently, the non-coding genome has gained prominence due to its critical role in development and disease. Here, we have re-purposed the Ac/Ds maize transposition system to reliably screen and efficiently characterise zebrafish enhancers, with or without germline propagation. We further utilised the system to stably express guide RNAs in microinjected embryos enabling tissue-specific CRISPR/dCas9-interference (CRISPRi) knockdown of lncRNA and enhancer activity without disrupting the underlying genetic sequence. Our study highlights the utility of Ac/Ds transposition for transient epigenome modulation of non-coding elements in zebrafish.\n\nSummary statementWe adapted the Ac/Ds transposition system, which enables continuous expression of guide RNAs for CRISPR/dCas9 perturbation, to examine the function of non-coding RNAs and enhancer elements in zebrafish.

developmental biology↗

bioRxiv2018

Transgenic Mice and Pluripotent Stem Cells Express EGFP under the Control of miR-302 Promoter

MicroRNAs are a group of short non-coding RNAs that undertake various roles in different cell signaling pathways and developmental stages. They regulate gene expression levels at the post-transcriptional stage, which results in cleavage of mRNAs or repression of their translation. Some miRNAs, including the miR-302 cluster, are critical regulators for the stemness state of embryonic stem cells and cell fate patterning. The miR-302 cluster is located in the intron of a non-coding gene that has no other reported function, other than hosting miR-302, and grant a complex expression regulation through upstream its regulatory sequences. To date, analysis of the miR-302 expression pattern in a transgenic mouse model has not been reported. In this study, we generated transgenic mice that expressed EGFP driven by miR-302 upstream regulatory sequences that harbored the core promoter of its host gene. We examined the activity of the miR-302 promotor in somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that miR-302 highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and has low expression in the somatic tissues of transgenic mice. It could be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages in mice.

developmental biology↗

bioRxiv2018

Ask1 and Akt act synergistically to promote ROS-dependent regeneration in Drosophila

The mechanism by which apoptotic cells release signals that induce undamaged neighbor cells to proliferate and regenerate missing parts remains elusive. Oxidative stress originated by dying or damaged cells can be propagated to neighboring cells, which then promote regeneration. We investigated the nature of the stress sensing mechanism by which neighboring cells are recruited. We found that Drosophila apoptosis signal-regulating kinase 1 (Ask1) senses reactive oxygen species (ROS) differently in stressed dying cells and unstressed neighboring cells and this differential sensing is pivotal for tissue repair. In undamaged cells, this activity is attenuated, but not abolished, by Akt1 phosphorylation, which thus acts as a survival signal that results in the tolerable levels of p38 and JNK necessary for regeneration. These observations demonstrate that the non-autonomous activation of the ROS-sensing mechanism by Ask1 and Akt1 in neighboring unstressed cells. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.\n\nAuthor summaryOne of the early events that occur after tissue damage is oxidative stress production that signals to initiate wound healing and regeneration. Several signaling pathways, such as JNK and p38, respond to oxidative stress and are necessary for regeneration. We decided to explore the mechanism that links the oxidative stress and the activation of these pathways. We used epithelia of Drosophila to genetically direct cell death in specific zones of the tissue as means of experimentally controlled cell damage. We found that the Ask1 protein, which is sensitive to oxidative stress, is a key player in this scenario. Actually it acts as an intracellular sensor that upon damage activates those signaling pathways. However, high activity of Ask1 can be toxic for the cell. This is controlled by Akt, an enzyme dowstream the insulin pathway, with attenuates the activity of Ask1 to tolerable levels. In conclusion, Ask1 and Akt act synergistically to respond to the stress generated after tissue damage and drive regeneration. In other words, we found that the link between oxidative stress and nutrition is key for tissue regeneration.

developmental biology↗

bioRxiv2018

Transitions in cell potency during early mouse development are driven by Notch

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naive pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

developmental biology↗

bioRxiv2018

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, but neither loss nor gain of hmx3a function was sufficient to generate ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

developmental biology↗

bioRxiv2018

Nuclear export in somatic cyst cells controls cyst cell-germline coordination and germline differentiation in the Drosophila testis

Nucleocytoplasmic communication is crucial for proper cell function and coordination of intrinsic cues with signaling responses emanating from the neighboring cells and the local tissue microenvironment. In the Drosophila male germline system, germ cells proliferate and progressively differentiate enclosed in supportive somatic cyst cells, forming a small cyst, the functional unit of differentiation. Here we show that the peripheral nucleoporins Nup62, Nup214 and Nup88, and the exportin Emb are critically required in cyst cells to maintain cyst cell survival and germline encapsulation in order to protect cyst cell-germline communication and promote germ cell differentiation. Knockdown of nup62, emb, nup214 or nup88 in cyst cells leads to cell-autonomous defects in mRNA export, and cell non-autonomous overproliferation of early germ cells in the absence of cyst cell-derived differentiation signals. Suppression of apoptosis can reverse cyst cell elimination and partially restored those defects. Interestingly, overexpression of the Drosophila Profilin gene chickadee can rescue cyst cell survival and restore germline encapsulation and differentiation, by counteracting Ntf-2 mediated export, suggesting that the function of Profilin in cyst cells is linked to nuclear export.

developmental biology↗