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bioRxiv2018

Simultaneous Deletion of Transient Receptor Potential Vanilloid 3 and Cacna1h Undermines Ca2+ Homeostasis in Oocytes and Fertility in Mice

In mammals, calcium (Ca2+) influx fills the endoplasmic reticulum, from where Ca2+ is released following fertilization to induce egg activation. However, an incomplete index of the plasma membrane channels and their specific contributions that underlie this influx in oocytes and eggs led us to simultaneously knock out the transient receptor potential vanilloid, member 3 (TRPV3) channel and the T-type channel, CaV3.2. Double knockout (dKO) females displayed subfertility and their oocytes and eggs showed significantly diminished Ca2+ store content and oscillations after fertilization compared to controls. We also found that the cell cycle stage during maturation determines the functional expression of channels whereby they show a distinct permeability to certain ions. In total, we demonstrate that TRPV3 and CaV3.2 are required for initiating physiological oscillations and that Ca2+ influx dictates the periodicity of oscillations during fertilization. dKO gametes will be indispensable to identify the complete native channel currents present in mammalian eggs.

developmental biology↗

bioRxiv2018

Nanopore formation in the cuticle of the insect olfactory sensillum

Insects sense odorants through small (50-200-nm diameter) pores in the cuticle covering the olfactory sensilla. These nanopores serve as a filter, preventing the entry of larger airborne particles and limiting water loss. Here we show that the nanopores in Drosophila originate from a curved ultrathin film formed in the outermost layer of the cuticle, secreted from specialized plasma membrane protrusions. The gore-tex/Osiris23 gene, encoding an endosomal protein, is required for nanopore formation and odor receptivity, and is expressed specifically in developing olfactory shaft cells. The 24-member Osiris gene family is expressed in various cuticle-secreting cells, and is found only in insect genomes. Some Osiris mutants show defective cuticle-associated functions, suggesting that Osiris genes may provide a platform for investigating functional surface nano-fabrication in insects.\n\nOne Sentence SummaryAn insect-specific endosomal protein promotes the formation of nano-scale pore structures of the apical extracellular matrix.

developmental biology↗

bioRxiv2018

MicroRNAs buffer genetic variation at specific temperatures during embryonic development

Successful embryogenesis requires the coordination of developmental events. Perturbations, such as environmental changes, must be buffered to ensure robust development. However, how such buffering occurs is currently unknown in most developmental systems. Here, we demonstrate that seven miRNAs are differentially expressed during Drosophila embryogenesis at varying temperatures within natural physiological ranges. Lack of miR-3-309, -31a, -310c, -980 or -984c causes developmental delays specifically at a given temperature. Detailed analysis on miR-310c and -984c shows that their targets are typically mis-expressed in mutant backgrounds, with phenotypes more pronounced at temperatures where miRNAs show highest expression in wild-type embryos. Our results show that phenotypes may arise at specific temperatures while remaining silent at others, even within typical temperature ranges. Our work uncovers that miRNAs mask genetic variation at specific temperatures to increase embryonic robustness, highlighting another layer of complexity in miRNA expression.

developmental biology↗

bioRxiv2018

ahr2, but not ahr1a or ahr1b, is required for craniofacial and fin development and TCDD-dependent cardiotoxicity in zebrafish

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds environmental toxins and regulates gene expression. AHR also regulates developmental processes, like craniofacial development and hematopoiesis, in the absence of environmental exposures. Zebrafish have three paralogues of AHR: ahr1a, ahr1b and ahr2. Adult zebrafish with mutations in ahr2 exhibited craniofacial and fin defects. However, the degree to which ahr1a and ahr1b influence ahr2 signaling and contribute to fin and craniofacial development are not known. We compared morphology of adult ahr2 mutants and ahr1a/ahr1b single and double mutant zebrafish. We found that ahr1a/ahr1b single and double mutants were morphologically normal while ahr2 mutant zebrafish demonstrated fin and craniofacial malformations. At 5 days post fertilization, both ahr1a/ahr1b and ahr2 mutant larvae were normal, suggesting that adult phenotypes are due to defects in maturation or maintenance. AHR was shown to interact with estrogen receptor alpha, yet it is not known whether these interactions are constitutive or dependent on ahr1 genes. To determine whether estrogen receptors are constitutive cofactors for AHR signaling, we used genetic and pharmacologic techniques to analyze TCDD-dependent toxicity in estrogen receptor and ahr mutant embryos. We found that embryos with mutations in ahr1a/ahr1b or estrogen receptor genes are susceptible to TCDD toxicity while ahr2 mutant embryos are TCDD-resistant. Moreover, pharmacologic blockade of nuclear estrogen receptors failed to prevent TCDD toxicity. These findings suggest that ahr1 genes do not have overlapping functions with ahr2 in fin and craniofacial development or TCDD-dependent toxicity, and that estrogen receptors are not constitutive partners of ahr2.

developmental biology↗

bioRxiv2018

Development of a robust automated tool for the annotation of embryo morphokinetic parameters

Study QuestionIs it possible to automatically annotate human embryo development in time-lapse devices, with results comparable to manual annotation?\n\nSummary AnswerWe developed an automated tool for the annotation of embryo morphokinetic parameters having a high concordance with expert manual annotation in a large scale-study.\n\nWhat is Known AlreadyMorphokinetic parameters obtained with time-lapse devices are increasingly used for human embryo quality assessment. However, their annotation is timeconsuming and can be operator-dependent, highlighting the need of developing automated approaches.\n\nStudy Design, Size, DurationThis monocentric pilot study was conducted using 701 blastocysts originating from 584 couples undergoing IVF with embryo culture in a time-lapse device and on 4 mouse embryos.\n\nParticipants/Materials, Setting, MethodsAn automated annotation tool was developed based on grey level coefficient of variation and detection of the thickness of the zona pellucida. The timings of cellular events obtained with the automated tool were compared with those obtained manually by 2 expert embryologists. The same procedure was applied on 4 mouse preimplantation embryos obtained with a different device in a different setting.\n\nMain Results and the Role of ChanceAlthough some differences were found when embryos were considered individually, we found an overall excellent concordance between automated and manual annotation of human embryo morphokinetics from fertilization to expanded blastocyst stage (r2=0.94). Moreover, the automated annotation tool gave promising results across species (human, mice).\n\nLimitations, Reasons for CautionThese results should undergo multi-centric external evaluation in order to test the overall performance of the annotation tool.\n\nWider Implications of the FindingsOur system performs significantly better than the ones reported in the literature and on a bigger cohort, paving the way for high-throughput analysis of multicentric morphokinetic databases, providing new insights into the clinical value of morphokinetics as predictor of embryo quality and implantation.\n\nStudy Funding/Competing Interest(s)This study was partly funded by Finox Forward Grant 2016.\n\nTrial Registration NumberNA

developmental biology↗

bioRxiv2018

Smed-egfr-4 is required for planarian eye regeneration

Planarians are amazing animals that can regenerate a whole body from a tiny piece of them thanks to their pluripotent stem cells, the neoblasts. Planarian neoblasts include both pluripotent stem cells and specialized lineage-committed progenitors that give rise to all the mature cell types during regeneration and homeostatic cell turnover in these plastic animals. Little is known, however, about the mechanisms that regulate neoblast differentiation. Recently, it has been shown that Smed-egfr-1, a homologue of the epidermal growth factor receptor (EGFR) family is required for the final differentiation of the gut progenitors into mature cells but not for their specification. As planarians have several EGFR homologues it has been proposed that they could have diverged functionally to regulate the differentiation of the different cell types found in these animals. Here, we report on the function of Smed-egfr-4 on eye regeneration. The silencing of this gene by RNAi results in animals regenerating smaller eyes compared to controls. The numbers of both eye mature cell types, photoreceptor neurons and eye-cup pigment cells, are significantly decreased in the Smed-egfr-4(RNAi) animals. In contrast, the number of eye progenitor cells expressing the specific markers Smed-ovo and Smed-sp6-9 is increased. These results suggest that Smed-egfr-4 would be required not for the specification of eye progenitor cells but rather for their final differentiation and support the idea that in planarians the EGFR pathway could play a general role regulating the differentiation of lineage-committed progenitors.

developmental biology↗

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

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↗