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Acute myeloid leukemia (AML) accounts for greater than twenty thousand new cases of leukemia annually in the United States. The average five-year survival rate is approximately 30%, pointing to the need for developing novel model systems for drug discovery. In particular, patients with chromosomal rearrangements in the mixed lineage leukemia (MLL) gene have higher relapse rates with poor outcomes. In this study we investigated the expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos. We observed an expansion of MLL positive cells and determined these cells colocalized with the myeloid markers spi1b, mpx, and mpeg. In addition, expression of MLL-ENL and MLL-AF9 induced the expression of endogenous bcl2 and cdk9, genes that are often dysregulated in MLL-r-AML. Co-treatment of lyz: MLL-ENL or lyz:MLL-AF9 expressing embryos with the BCL2 inhibitor, Venetoclax, and the CDK9 inhibitor, Flavopiridol, significantly reduced the number of MLL positive cells compared to embryos treated with vehicle or either drug alone. In addition, cotreatment with Venetoclax and Flavopiridol significantly reduced the expression of endogenous mcl1a compared to vehicle, consist with AML. This new model of MLL-r-AML provides a novel tool to understand the molecular mechanisms underlying disease progression and a platform for drug discovery. Author summary: Acute myeloid leukemia (AML) accounts for over twenty thousand new cases of leukemia per year in the United States. MLL rearrangements (MLL-r) occur in approximately 10% of AML patients, with 34% of those harboring an MLL-ENL or MLL-AF9 rearrangement. Patients with an MLL-r have a higher relapse rate and their overall survival is low, pointing to a need for novel therapeutic interventions. Zebrafish have emerged as a powerful model system to study hematological malignancies due their conserved hematopoietic program, genetic tractability, and pharmacological accessibility. Expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos induced an expansion of myeloid cells and caused an increase in expression of endogenous bcl2 and cdk9. MLL expression was significantly reduced in lyz:MLL-ENL and lyz-MLL-AF9 embryos co-treated with the BCL2 inhibitor (Venetoclax) and the CDK9 inhibitor (Flavopiridol) compared to embryos treated with vehicle or either drug alone. Reduced MLL expression occurred in conjunction with a significant decrease in mcl1a expression, consistent with human AML cells. Taken together, these results further support the use of zebrafish to model hematological malignancies for drug discovery. [ABSTRACT FROM AUTHOR]

How gene regulatory networks (GRNs) encode gene expression dynamics and how GRNs evolve are not well understood, although these problems have been studied extensively. We created a digital twin that accurately reproduces expression dynamics of 13 genes that initiate expression in 32-cell ascidian embryos. We first showed that gene expression patterns can be manipulated according to predictions by this digital model. Next, to simulate GRN rewiring, we changed regulatory functions that represented their regulatory mechanisms in the digital twin, and found that in 55 of 100 cases, removal of a single regulator from a conjunctive clause of Boolean functions did not theoretically alter qualitative expression patterns of these genes. In other words, we found that more than half the regulators gave theoretically redundant temporal or spatial information to target genes. We experimentally substantiated that the expression pattern of Nodal was maintained without one of these factors, Zfpm, by changing the upstream regulatory sequence of Nodal. Such robust buffers of regulatory mechanisms may provide a basis of enabling developmental system drift, or rewiring of GRNs without changing expression patterns of downstream genes, during evolution. Author summary: Although regulatory relationships in gene regulatory networks have been studied extensively during the past two decades, it is not fully understood how gene regulatory networks produce gene expression dynamics. One major reason is the difficulty of experimentally determining "regulatory functions" that mathematically describe how individual genes are regulated. Because of simplicity of embryonic structure and compactness of the genome, ascidian embryos provide an ideal system for analyzing dynamics of gene regulatory networks. In 32-cell ascidian embryos, there are only 13 regulatory genes that initiate expression. Regulatory functions for these genes, which are represented as Boolean functions, collectively constitute a digital twin that reproduces gene expression dynamics at single-cell resolution throughout the whole embryo. The digital twin is a powerful tool for exploring mechanisms that are not readily accessible in experiments using real embryos. We found that over half the regulators in regulatory functions are potentially redundant for specifying temporal and spatial gene expression patterns. It is generally believed that regulatory functions have changed frequently during evolution, and our results show that a gene regulatory network in early ascidian embryos has robust buffers, which constitute a basis for developmental system drift. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are ubiquitous organelles that facilitate neutral lipid storage in cells, including energy-dense triglycerides. They are found in all investigated metazoan embryos where they are thought to provide energy for development. Intriguingly, early embryos of diverse metazoan species asymmetrically allocate LDs amongst cellular lineages, a process which can involve massive intracellular redistribution of LDs. However, the biological reason for asymmetric lineage allocation is unknown. To address this issue, we utilize the Drosophila embryo where the cytoskeletal mechanisms that drive allocation are well characterized. We disrupt allocation by two different means: Loss of the LD protein Jabba results in LDs adhering inappropriately to glycogen granules; loss of Klar alters the activities of the microtubule motors that move LDs. Both mutants cause the same dramatic change in LD tissue inheritance, shifting allocation of the majority of LDs to the yolk cell instead of the incipient epithelium. Embryos with such mislocalized LDs do not fully consume their LDs and are delayed in hatching. Through use of a dPLIN2 mutant, which appropriately localizes a smaller pool of LDs, we find that failed LD transport and a smaller LD pool affect embryogenesis in a similar manner. Embryos of all three mutants display overlapping changes in their transcriptome and proteome, suggesting that lipid deprivation results in a shared embryonic response and a widespread change in metabolism. Excitingly, we find abundant changes related to redox homeostasis, with many proteins related to glutathione metabolism upregulated. LD deprived embryos have an increase in peroxidized lipids and rely on increased utilization of glutathione-related proteins for survival. Thus, embryos are apparently able to mount a beneficial response upon lipid stress, rewiring their metabolism to survive. In summary, we demonstrate that early embryos allocate LDs into specific lineages for subsequent optimal utilization, thus protecting against oxidative stress and ensuring punctual development. Author summary: Embryos of diverse animal species sort their lipid droplets into specific cell lineages early in development. Here we prevent Drosophila embryo's ability to undergo this asymmetric inheritance through two separate genetic perturbations. We then investigate the effects on subsequent embryogenesis, finding developmental delays and an inability to consume the mislocalized lipid droplets. We find a similar delay for embryos which receive fewer lipid droplets from their mothers. To understand how embryos respond to limited access to lipid droplets, we investigate the global transcriptome and proteome in the mutants and find alterations to the expression of hundreds of metabolism genes, including for enzymes that transport sugar across cell membranes, break down fat, or protect against oxidative stress. We test if the upregulated genes have beneficial functions for lipid-deprived embryos. We find that zygotic knock down of the upregulated oxidative stress response genes GSS and GSTT4 as well as the lipase ATGL lowers hatching success in LD-deprived genetic backgrounds, but not wild type. In summary, we find that early lipid droplet lineage sorting sets the stage for metabolic success in subsequent embryogenesis. [ABSTRACT FROM AUTHOR]

Aberrantly up-regulated activity of the type II transmembrane protease Matriptase-1 has been associated with the development and progression of a range of epithelial-derived carcinomas, and a variety of signaling pathways can mediate Matriptase-dependent tumorigenic events. During mammalian carcinogenesis, gain of Matriptase activity often results from imbalanced ratios between Matriptase and its cognate transmembrane inhibitor Hai1. Similarly, in zebrafish, unrestrained Matriptase activity due to loss of hai1a results in epidermal pre-neoplasms already during embryogenesis. Here, based on our former findings of a similar tumor-suppressive role for the Na+/K+-pump beta subunit ATP1b1a, we identify epithelial polarity defects and systemic hypotonic stress as another mode of aberrant Matriptase activation in the embryonic zebrafish epidermis in vivo. In this case, however, a different oncogenic pathway is activated which contains PI3K, AKT and NFkB, rather than EGFR and PLD (as in hai1a mutants). Strikingly, epidermal pre-neoplasm is only induced when epithelial polarity defects in keratinocytes (leading to disturbed Matriptase subcellular localization) occur in combination with systemic hypotonic stress (leading to increased proteolytic activity of Matriptase). A similar combinatorial effect of hypotonicity and loss of epithelial polarity was also obtained for the activity levels of Matriptase-1 in human MCF-10A epithelial breast cells. Together, this is in line with the multi-factor concept of carcinogenesis, with the notion that such factors can even branch off from one and the same initiator (here ATP1a1b) and can converge again at the level of one and the same mediator (here Matriptase). In sum, our data point to tonicity and epithelial cell polarity as evolutionarily conserved regulators of Matriptase activity that upon de-regulation can constitute an alternative mode of Matriptase-dependent carcinogenesis in vivo. Author summary: Deciphering the mechanisms of carcinogenesis is pivotal not only for the treatment of the disease, but also for identifying potential risk factors. Model systems can help to achieve this goal. Work with zebrafish has identified several evolutionarily conserved factors driving pre-neoplastic events in the epidermis already at embryonic stages. This study focuses on the oncogene ST14, encoding the type II transmembrane protease Matriptase-1 that upon hyperactivation—as for instance due to genetic loss of its cognate transmembrane inhibitor Hai1—can drive different carcinomas. Here, we identify two additional conditions promoting oncogenic Matriptase-1 hyperactivation: hypotonic stress and compromised epithelial cell polarity. Of note, these two conditions need to occur together for Matriptase-dependent induction of pre-neoplasms in the embryonic zebrafish epidermis, in line with the multi-factor concept of carcinogenesis. Hypotonicity increases the proteolytic activity of Matriptase, while epithelial polarity defects lead to a subcellular mis-localization of Matriptase in outer epidermal cells, bringing more of it into close contact to underlying basal cells and thus inducing oncogenic pathways in them. Together, our work identifies novel modes of oncogenic Matriptase hyperactivation and function in vivo, originating from cells of the tumor environment, rather than from the tumor cells themselves. [ABSTRACT FROM AUTHOR]

Embryogenesis requires coordinated gene regulatory activities early on that establish the trajectory of subsequent development, during a period called the maternal-to-zygotic transition (MZT). The MZT comprises transcriptional activation of the embryonic genome and post-transcriptional regulation of egg-inherited maternal mRNA. Investigation into the MZT in animals has focused almost exclusively on bilaterians, which include all classical models such as flies, worms, sea urchin, and vertebrates, thus limiting our capacity to understand the gene regulatory paradigms uniting the MZT across all animals. Here, we elucidate the MZT of a non-bilaterian, the cnidarian Hydractinia symbiolongicarpus. Using parallel poly(A)-selected and non poly(A)-dependent RNA-seq approaches, we find that the Hydractinia MZT is composed of regulatory activities similar to many bilaterians, including cytoplasmic readenylation of maternally contributed mRNA, delayed genome activation, and separate phases of maternal mRNA deadenylation and degradation that likely depend on both maternally and zygotically encoded clearance factors, including microRNAs. But we also observe massive upregulation of histone genes and an expanded repertoire of predicted H4K20 methyltransferases, aspects thus far particular to the Hydractinia MZT and potentially underlying a novel mode of early embryonic chromatin regulation. Thus, similar regulatory strategies with taxon-specific elaboration underlie the MZT in both bilaterian and non-bilaterian embryos, providing insight into how an essential developmental transition may have arisen in ancestral animals. Author summary: Eggs are preloaded with genetic instructions encoded as mRNA, which not only drive the first stages of embryogenesis, but also induce the production of new mRNA from the embryonic genome that supplant the egg-inherited ones. Many different species undergo this process, but the extent to which they share common mechanisms to regulate the transition from egg to embryonic mRNA is still unclear, especially among very distant animal relatives to humans. We have investigated this transition in Hydractinia, a representative of the Cnidaria phylum that also includes jellyfish and sea anemones. Using optimized methods to measure mRNA quantities during early Hydractinia development, we find an overall trajectory of mRNA change closely resembling what occurs in embryos of more commonly studied animals, as well as evidence for similar mechanisms regulating these changes. However, we also uncovered surprising patterns of gene expression for chromosome structural genes (histones) and enzymes that chemically modify them, suggesting that Hydractinia embryos regulate their genomes in a novel way compared to what has previously been observed. Our findings provide insight into how our common animal ancestor may have regulated early development, but also reveal a new aspect of gene regulation that warrants further investigation in other contexts. [ABSTRACT FROM AUTHOR]

Histones are essential for chromatin packaging, and histone supply must be tightly regulated as excess histones are toxic. To drive the rapid cell cycles of the early embryo, however, excess histones are maternally deposited. Therefore, soluble histones must be buffered by histone chaperones, but the chaperone necessary to stabilize soluble H3-H4 pools in the Drosophila embryo has yet to be identified. Here, we show that CG8223, the Drosophila homolog of NASP, is a H3-H4-specific chaperone in the early embryo. We demonstrate that, while a NASP null mutant is viable in Drosophila, NASP is maternal effect gene. Embryos laid by NASP mutant mothers have a reduced rate of hatching and show defects in early embryogenesis. Critically, soluble H3-H4 pools are degraded in embryos laid by NASP mutant mothers. Our work identifies NASP as the critical H3-H4 histone chaperone in the Drosophila embryo. Author summary: Histones H2A, H2B, H3, and H4 are small proteins required for the basic unit of DNA packaging. Overexpression or depletion of histones is toxic to cells, therefore histone synthesis, trafficking, storage and deposition into chromatin requires careful regulation. Histone chaperones influence nearly all aspects of histone metabolism, including histone storage. During Drosophila development, excess histones are maternally provided to the early embryo to drive the early rapid nuclear cycles. To prevent histone degradation, maternal deposits of histones must be stabilized by a histone chaperone. To date, however, the histone chaperone that stabilizes H3-H4 in the early embryo has remained unknown. Based on sequence, structure and function, we show that the Drosophila homolog of NASP is the critical H3-H4 histone chaperone in the Drosophila early embryo. NASP is required to stabilize the maternally deposited H3-H4 supply that fuels early embryogenesis. While NASP is not an essential gene, embryos laid by NASP mutant mothers show several defects in embryogenesis. Our work defines fundamental aspects of H3 and H4 storage and stability in the early Drosophila embryo. [ABSTRACT FROM AUTHOR]

Overexpression of the TGFβ pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFβ promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFβ signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFβ pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFβ and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFβ-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFβ-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFβ-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis. Author summary: The TGFβ pathway inhibits the growth and proliferation of the hematopoietic stem and progenitor cells in Fanconi anemia (FA). We have previously shown that inhibition of the TGFβ pathway rescues the growth defects of FA hematopoietic stem and progenitor cells. In this work we determined whether a cross between Fancd2 (an FA gene) and Smad3 (the canonical TGFβ pathway transducer) heterozygous mice would generate double knockout (Fancd2-/-Smad3-/-) progeny without the hematopoietic defect characteristic of FA. Contrary to expected, we observed increased embryonic lethality for the double knockout (DKO) mice and persistence of the FA phenotype in the surviving DKO mice. We discovered that Fancd2-/- embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. In addition, the Fancd2-/- mouse embryos exhibited high NHEJ activity which allowed them to survive during gestation. The few DKO survivors retain the FA phenotype due to activation of the non-canonical TGFβ -ERK pathway. [ABSTRACT FROM AUTHOR]

The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice. Author summary: The first and second cell fate decisions of a new life are important for subsequent embryonic and placental development. These events are finely controlled by a network of transcriptional factors, which are extensively characterized in mice. Species-specific roles of these proteins are emerging in mammals. Here, we develop a gene loss-of-function system by using cytosine base editors in bovine early embryos. We find that expression pattern, functional roles, and regulation of SOX2 are all different between mouse and bovine embryos. Remarkably, SOX2 is extremely important for OCT4 and NANOG, two well-established pluripotency factors. Furthermore, CDX2 is required to repress SOX2 in the trophectoderm. Given similar expression pattern of SOX2 between human and bovine blastocysts, bovine embryo represents a putative model to investigate human pluripotency regulation in vivo. [ABSTRACT FROM AUTHOR]

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation. Author summary: As aquatic organisms, fish must tolerate environmental challenges that include acid-base fluctuations and water-borne pathogens. The skin provides a first line of defense against these challenges, and specific cell types in the tissue are responsible for different protective functions. For example, keratinocytes provide body coverage, ionocytes are responsible for maintaining body fluid ionic homeostasis, and epidermal mucous cells generate a protective layer of mucus that covers the entire fish surface. In this study, we uncovered the developmental process in zebrafish that underlies the generation of epidermal mucous cells. First, we characterized epidermal mucous cells according to their expression of a particular gene, agr2. Then, we found that these cells differentiate soon after ionocytes and keratinocytes, and the molecular pathways that guide differentiation of all three cell types involve similar signals. While ionocytes and keratinocytes are known to be specified by Delta-Notch-mediated lateral inhibition, we found that epidermal mucous cells are specified by activation of Notch by Delta and Jagged ligands. Thus, our results suggest that the specification of these major cell types in the epidermis occurs via a streamlined Notch-dependent process. This utilization of temporally distinct signaling events can therefore generate diverse cell types in the fish epidermis. [ABSTRACT FROM AUTHOR]

EHMT2 is the main euchromatic H3K9 methyltransferase. Embryos with zygotic, or maternal mutation in the Ehmt2 gene exhibit variable developmental delay. To understand how EHMT2 prevents variable developmental delay we performed RNA sequencing of mutant and somite stage-matched normal embryos at 8.5–9.5 days of gestation. Using four-way comparisons between delayed and normal embryos we clarified what it takes to be normal and what it takes to develop. We identified differentially expressed genes, for example Hox genes that simply reflected the difference in developmental progression of wild type and the delayed mutant uterus-mate embryos. By comparing wild type and zygotic mutant embryos along the same developmental window we detected a role of EHMT2 in suppressing variation in the transcriptional switches. We identified transcription changes where precise switching during development occurred only in the normal but not in the mutant embryo. At the 6-somite stage, gastrulation-specific genes were not precisely switched off in the Ehmt2−/− zygotic mutant embryos, while genes involved in organ growth, connective tissue development, striated muscle development, muscle differentiation, and cartilage development were not precisely switched on. The Ehmt2mat−/+ maternal mutant embryos displayed high transcriptional variation consistent with their variable survival. Variable derepression of transcripts occurred dominantly in the maternally inherited allele. Transcription was normal in the parental haploinsufficient wild type embryos despite their delay, consistent with their good prospects. Global profiling of transposable elements revealed EHMT2 targeted DNA methylation and suppression at LTR repeats, mostly ERVKs. In Ehmt2−/− embryos, transcription over very long distances initiated from such misregulated 'driver' ERVK repeats, encompassing a multitude of misexpressed 'passenger' repeats. In summary, EHMT2 reduced transcriptional variation of developmental switch genes and developmentally switching repeat elements at the six-somite stage embryos. These findings establish EHMT2 as a suppressor of transcriptional and developmental variation at the transition between gastrulation and organ specification. Author summary: Developmental variation is the property of normal development, and its regulation is poorly understood. Variable developmental delay is found in embryos that carry mutations of epigenetic modifiers, suggesting a role of chromatin in controlling developmental delay and its variable nature. We analyzed a genetic series of mutations and found that EHMT2 suppresses variation of developmental delay and also suppresses the variation of transcriptional switches at the transition between gastrulation and organ specification. [ABSTRACT FROM AUTHOR]

Studies in various animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are scarce and limited to a handful of model organisms. In cephalochordates (amphioxus), an early branching chordate group, maternal transcripts of germline determinants form a compact granule that is inherited by a single blastomere during cleavage stages. Further blastomere separation experiments suggest that other transcripts associated with the granule are likely responsible for organizing the posterior structure in amphioxus; however, the identities of these determinants remain unknown. In this study, we used high-throughput RNA sequencing of separated blastomeres to examine asymmetrically localized transcripts in two-cell and eight-cell stage embryos of the amphioxus Branchiostoma floridae. We identified 111 and 391 differentially enriched transcripts at the 2-cell stage and the 8-cell stage, respectively, and used in situ hybridization to validate the spatial distribution patterns for a subset of these transcripts. The identified transcripts could be categorized into two major groups: (1) vegetal tier/germ granule-enriched and (2) animal tier/anterior-enriched transcripts. Using zebrafish as a surrogate model system, we showed that overexpression of one animal tier/anterior-localized amphioxus transcript, zfp665, causes a dorsalization/anteriorization phenotype in zebrafish embryos by downregulating the expression of the ventral gene, eve1, suggesting a potential function of zfp665 in early axial patterning. Our results provide a global transcriptomic blueprint for early-stage amphioxus embryos. This dataset represents a rich platform to guide future characterization of molecular players in early amphioxus development and to elucidate conservation and divergence of developmental programs during chordate evolution. Author summary: Studies in model animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are limited to a handful of organisms. Here, we use a PCR-based single-cell RNA sequencing approach on separated blastomeres from the cleavage stage embryos of amphioxus, an early-branching chordate. We generate a spatial transcriptomic map and demonstrate the mosaic property of the early amphioxus embryo, contrasting to the general notion that its embryogenesis is highly regulative. Our cross-species experiments further provide evidence to support that some of the identified factors play functions in early axial patterning. Our results provide a global transcriptomic blueprint that can serve as a rich platform for guiding future characterization of molecular players in early amphioxus development and for comparative studies on testing conservation and divergence of developmental programs during chordate evolution. [ABSTRACT FROM AUTHOR]

During metazoan development, the cell cycle is remodelled to coordinate proliferation with differentiation. Developmental cues cause dramatic changes in the number and timing of replication initiation events, but the mechanisms and physiological importance of such changes are poorly understood. Cyclin-dependent kinases (CDKs) are important for regulating S-phase length in many metazoa, and here we show in the nematode Caenorhabditis elegans that an essential function of CDKs during early embryogenesis is to regulate the interactions between three replication initiation factors SLD-3, SLD-2 and MUS-101 (Dpb11/TopBP1). Mutations that bypass the requirement for CDKs to generate interactions between these factors is partly sufficient for viability in the absence of Cyclin E, demonstrating that this is a critical embryonic function of this Cyclin. Both SLD-2 and SLD-3 are asymmetrically localised in the early embryo and the levels of these proteins inversely correlate with S-phase length. We also show that SLD-2 asymmetry is determined by direct interaction with the polarity protein PKC-3. This study explains an essential function of CDKs for replication initiation in a metazoan and provides the first direct molecular mechanism through which polarization of the embryo is coordinated with DNA replication initiation factors. Author summary: How and when a cell divides changes as the cell assumes different fates. How these changes in cell division are brought about are poorly understood, but are critical to ensure that cells do not over-proliferate leading to cancer. The nematode C. elegans is an excellent system to study the role of cell cycle changes during animal development. Here we show that two factors SLD-2 and SLD-3 are critical to control the decision to begin genome duplication. We show that these factors are differently distributed to different cell lineages in the early embryo, which may be a key event in determining the cell cycle rate in these cells. For the first time we show that, PKC-3, a key component of the machinery that determines the front (anterior) from the back (posterior) of the embryo directly controls SLD-2 distribution, which might explain how the polarisation of the embryo causes changes in the proliferation of different cell lineages. As PKC-3 is frequently mutated in human cancers, how this factor controls cell proliferation may be important to understand tumour progression. [ABSTRACT FROM AUTHOR]

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3-4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells.

Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ release channels located on the ER membrane, which in mammals consist of 3 different subtypes (IP3R1, IP3R2, and IP3R3) encoded by 3 genes, Itpr1, Itpr2, and Itpr3, respectively. Studies utilizing genetic knockout mouse models have demonstrated that IP3Rs are essential for embryonic survival in a redundant manner. Deletion of both IP3R1 and IP3R2 has been shown to cause cardiovascular defects and embryonic lethality. However, it remains unknown which cell types account for the cardiovascular defects in IP3R1 and IP3R2 double knockout (DKO) mice. In this study, we generated conditional IP3R1 and IP3R2 knockout mouse models with both genes deleted in specific cardiovascular cell lineages. Our results revealed that deletion of IP3R1 and IP3R2 in cardiomyocytes by TnT-Cre, in endothelial / hematopoietic cells by Tie2-Cre and Flk1-Cre, or in early precursors of the cardiovascular lineages by Mesp1-Cre, resulted in no phenotypes. This demonstrated that deletion of both IP3R genes in cardiovascular cell lineages cannot account for the cardiovascular defects and embryonic lethality observed in DKO mice. We then revisited and performed more detailed phenotypic analysis in DKO embryos, and found that DKO embryos developed cardiovascular defects including reduced size of aortas, enlarged cardiac chambers, as well as growth retardation at embryonic day (E) 9.5, but in varied degrees of severity. Interestingly, we also observed allantoic-placental defects including reduced sizes of umbilical vessels and reduced depth of placental labyrinth in DKO embryos, which could occur independently from other phenotypes in DKO embryos even without obvious growth retardation. Furthermore, deletion of both IP3R1 and IP3R2 by the epiblast-specific Meox2-Cre, which targets all the fetal tissues and extraembryonic mesoderm but not extraembryonic trophoblast cells, also resulted in embryonic lethality and similar allantoic-placental defects. Taken together, our results demonstrated that IP3R1 and IP3R2 play an essential and redundant role in maintaining the integrity of fetal-maternal connection and embryonic viability. Author summary: Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ release channels located on the ER membrane, which in mammals consist of 3 different subtypes (IP3R1, IP3R2, and IP3R3). IP3R-mediated Ca2+ signaling has been proposed to play an essential role in regulating cardiovascular development, and IP3R1 and IP3R2 double knockout (DKO) mice has been shown to develop cardiovascular defects and embryonic lethality. However, our present study using conditional cell-specific gene deletion strategy revealed that deletion of both genes in cardiomyocytes, endothelial / hematopoietic cells, and early precursors of the cardiovascular lineages in mice could not result in similar lethal phenotypes. By contrast, we observed allantoic-placental defects including reduced sizes of umbilical vessels and reduced depth of placental labyrinth in DKO embryos, which could occur independently from other phenotypes in DKO embryos. We further found that deletion of both IP3R1 and IP3R2 in epiblast also resulted in embryonic lethality and similar allantoic-placental defects. Our results demonstrated that IP3R1 and IP3R2 play an essential and redundant role in maintaining the integrity of fetal-maternal connection and embryonic viability. [ABSTRACT FROM AUTHOR]

The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling. Author summary: Embryonic development starts with a series of fast nuclear divisions in most animals, which is followed by a dramatical cell cycle slowdown to enter a pause. Drosophila embryos undergo 13 fast and synchronous nuclear cycles with only S and M phases. In interphase 14, the cell cycle is remodeled: the mitosis pauses, the S phase is prolonged, and a gap phase is introduced. Post-translational regulation of Cdc25/Twine phosphatase is responsible for this remodeling. Although tribbles is involved, it has remained unclear how the timely degradation of Twine in interphase 14 is controlled. Here, we show that Protein phosphatase V (PpV) tightly controls Twine dynamics and thus the timing of cell cycle remodeling. PpV ensures appropriately low Twine levels at the onset of interphase 14 with little embryo-to-embryo variation. During interphase 14, tribbles and other factors are involved in the swift Twine degradation. This study provides insights in post-translational and safeguarding mechanisms of Cdc25, as well as in developmental timing of early embryogenesis. [ABSTRACT FROM AUTHOR]

The gene products that drive early development are critical for setting up developmental trajectories in all animals. The earliest stages of development are fueled by maternally provided mRNAs until the zygote can take over transcription of its own genome. In early development, both maternally deposited and zygotically transcribed gene products have been well characterized in model systems. Previously, we demonstrated that across the genus Drosophila, maternal and zygotic mRNAs are largely conserved but also showed a surprising amount of change across species, with more differences evolving at the zygotic stage than the maternal stage. In this study, we use comparative methods to elucidate the regulatory mechanisms underlying maternal deposition and zygotic transcription across species. Through motif analysis, we discovered considerable conservation of regulatory mechanisms associated with maternal transcription, as compared to zygotic transcription. We also found that the regulatory mechanisms active in the maternal and zygotic genomes are quite different. For maternally deposited genes, we uncovered many signals that are consistent with transcriptional regulation at the level of chromatin state through factors enriched in the ovary, rather than precisely controlled gene-specific factors. For genes expressed only by the zygotic genome, we found evidence for previously identified regulators such as Zelda and GAGA-factor, with multiple analyses pointing toward gene-specific regulation. The observed mechanisms of regulation are consistent with what is known about regulation in these two genomes: during oogenesis, the maternal genome is optimized to quickly produce a large volume of transcripts to provide to the oocyte; after zygotic genome activation, mechanisms are employed to activate transcription of specific genes in a spatiotemporally precise manner. Thus the genetic architecture of the maternal and zygotic genomes, and the specific requirements for the transcripts present at each stage of embryogenesis, determine the regulatory mechanisms responsible for transcripts present at these stages. Author summary: Early development in animals is a unique period of time, as it is controlled by gene products from two different genomes: that of the mother and that of the zygote. The earliest stages of development are directed by maternal mRNAs and proteins that are deposited into the egg, and only later does the zygote take over the transcription of its own genome. In this paper, we use data from 11 fruit fly species characterizing all the genes transcribed by the mother and by the zygote, to investigate how transcription is regulated in the maternal and zygotic genomes. While we find some conserved regulatory elements at both stages, regulation of maternal transcription is much more highly conserved across species. We present evidence that maternal transcription is controlled in large co-regulated chromatin domains, while zygotic transcription is much more gene-specific. These results make sense in the context of where these genes are being transcribed, as maternal transcripts are generated in support cells which churn out a large amount of mRNA during oogenesis, while zygotic genes are often transcribed in a particular time and place in the embryo. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA’s 3’ UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs interact to regulate gene expression is likely extensive, we have only begun to unravel the mechanisms of this functional cooperation. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK-1, in modulating miRNA activity during C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 plays a role in processing of some but not all miRNAs and is not required for ALG-1/AIN-1 miRISC assembly. We suggest that HRPK-1 may functionally interact with miRNAs by both affecting miRNA processing and by enhancing miRNA/miRISC gene regulatory activity and present models for its activity. [ABSTRACT FROM AUTHOR]

Comprehensive information on the timing and location of gene expression is fundamental to our understanding of embryonic development and tissue formation. While high-throughput in situ hybridization projects provide invaluable information about developmental gene expression patterns for model organisms like Drosophila, the output of these experiments is primarily qualitative, and a high proportion of protein coding genes and most non-coding genes lack any annotation. Accurate data-centric predictions of spatio-temporal gene expression will therefore complement current in situ hybridization efforts. Here, we applied a machine learning approach by training models on all public gene expression and chromatin data, even from whole-organism experiments, to provide genome-wide, quantitative spatio-temporal predictions for all genes. We developed structured in silico nano-dissection, a computational approach that predicts gene expression in >200 tissue-developmental stages. The algorithm integrates expression signals from a compendium of 6,378 genome-wide expression and chromatin profiling experiments in a cell lineage-aware fashion. We systematically evaluated our performance via cross-validation and experimentally confirmed 22 new predictions for four different embryonic tissues. The model also predicts complex, multi-tissue expression and developmental regulation with high accuracy. We further show the potential of applying these genome-wide predictions to extract tissue specificity signals from non-tissue-dissected experiments, and to prioritize tissues and stages for disease modeling. This resource, together with the exploratory tools are freely available at our webserver , which provides a valuable tool for a range of applications, from predicting spatio-temporal expression patterns to recognizing tissue signatures from differential gene expression profiles. [ABSTRACT FROM AUTHOR]

During vertebrate early embryogenesis, the ventral development is directed by the ventral-to-dorsal activity gradient of the bone morphogenetic protein (BMP) signaling. As secreted ligands, the extracellular traffic of BMP has been extensively studied. However, it remains poorly understood that how BMP ligands are secreted from BMP-producing cells. In this work, we show the dominant role of Marcksb controlling the secretory process of Bmp2b via interaction with Hsp70 in vivo. We firstly carefully characterized the role of Marcksb in promoting BMP signaling during dorsoventral axis formation through knockdown approach. We then showed that Marcksb cell autonomously regulates the trafficking of Bmp2b from producing cell to the extracellular space and both the total and the extracellular Bmp2b was decreased in Marcksb-deficient embryos. However, neither the zygotic mutant of marcksb (Zmarcksb) nor the maternal zygotic mutant of marcksb (MZmarcksb) showed any defects of dorsalization. In contrast, the MZmarcksb embryos even showed increased BMP signaling activity as measured by expression of BMP targets, phosphorylated Smad1/5/9 levels and imaging of Bmp2b, suggesting that a phenomenon of “genetic over-compensation” arose. Finally, we revealed that the over-compensation effects of BMP signaling in MZmarcksb was achieved through a sequential up-regulation of MARCKS-family members Marcksa, Marcksl1a and Marcksl1b, and MARCKS-interacting protein Hsp70.3. We concluded that the Marcksb modulates BMP signaling through regulating the secretory pathway of Bmp2b. [ABSTRACT FROM AUTHOR]

Animal development requires the execution of specific transcriptional programs in different sets of cells to build tissues and functional organs. Transcripts are exported from the nucleus to the cytoplasm where they are translated into proteins that, ultimately, carry out the cellular functions. Here we show that in Caenorhabditis elegans, reduction of mRNA export strongly affects epithelial morphogenesis and germline proliferation while other tissues remain relatively unaffected. Epithelialization and gamete formation demand a large number of transcripts in the cytoplasm for the duration of these processes. In addition, our findings highlight the existence of a regulatory feedback mechanism that activates gene expression in response to low levels of cytoplasmic mRNA. We expand the genetic characterization of nuclear export factor NXF-1 to other members of the mRNA export pathway to model mRNA export and recycling of NXF-1 back to the nucleus. Our model explains how mutations in genes involved in general processes, such as mRNA export, may result in tissue-specific developmental phenotypes. [ABSTRACT FROM AUTHOR]

We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared. [ABSTRACT FROM AUTHOR]

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases. [ABSTRACT FROM AUTHOR]

C-to-U editing is an important event in post-transcriptional RNA processing, which converts a specific cytidine (C)-to-uridine (U) in transcripts of mitochondria and plastids. Typically, the pentatricopeptide repeat (PPR) protein, which specifies the target C residue by binding to its upstream sequence, is involved in the editing of one or a few sites. Here we report a novel PPR-DYW protein EMP21 that is associated with editing of 81 sites in maize. EMP21 is localized in mitochondria and loss of the EMP21 function severely inhibits the embryogenesis and endosperm development in maize. From a scan of 35 mitochondrial transcripts produced by the Emp21 loss-of-function mutant, the C-to-U editing was found to be abolished at five sites (nad7-77, atp1-1292, atp8-437, nad3-275 and rps4-870), while reduced at 76 sites in 21 transcripts. In most cases, the failure to editing resulted in the translation of an incorrect residue. In consequence, the mutant became deficient with respect to the assembly and activity of mitochondrial complexes I and V. As six of the decreased editing sites in emp21 overlap with the affected editing sites in emp5-1, and the editing efficiency at rpl16-458 showed a substantial reduction in the emp21-1 emp5-4 double mutant compared with the emp21-1 and emp5-4 single mutants, we explored their interaction. A yeast two hybrid assay suggested that EMP21 does not interact with EMP5, but both EMP21 and EMP5 interact with ZmMORF8. Together, these results indicate that EMP21 is a novel PPR-DYW protein required for the editing of ~17% of mitochondrial target Cs, and the editing process may involve an interaction between EMP21 and ZmMORF8 (and probably other proteins). [ABSTRACT FROM AUTHOR]

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis for a variety of cancers, and promotes cell migration, invasion and metastasis. Little is known about its physiological role. In this study, we used mice with a gene-trap inactivated ASAP1 locus to study the functional role of ASAP1 in vivo, and found defects in tissues derived from mesenchymal progenitor cells. Loss of ASAP1 led to growth retardation and delayed ossification typified by enlarged hypertrophic zones in growth plates and disorganized chondro-osseous junctions. Furthermore, loss of ASAP1 led to delayed adipocyte development and reduced fat depot formation. Consistently, deletion of ASAP1 resulted in accelerated chondrogenic differentiation of mesenchymal cells in vitro, but suppressed osteo- and adipogenic differentiation. Mechanistically, we found that FAK/Src and PI3K/AKT signaling is compromised in Asap1GT /GT MEFs, leading to impaired adipogenic differentiation. Dysregulated FAK/Src and PI3K/AKT signaling is also associated with attenuated osteogenic differentiation. Together these observations suggest that ASAP1 plays a decisive role during the differentiation of mesenchymal progenitor cells. [ABSTRACT FROM AUTHOR]

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products. [ABSTRACT FROM AUTHOR]

Genomic imprinting in mammals was discovered over 30 years ago through elegant embryological and genetic experiments in mice. Imprinted genes show a monoallelic and parent of origin–specific expression pattern; the development of techniques that can distinguish between expression from maternal and paternal chromosomes in mice, combined with high-throughput strategies, has allowed for identification of many more imprinted genes, most of which are conserved in humans. Undoubtedly, technical progress has greatly promoted progress in the field of genomic imprinting. Here, we summarize the techniques used to discover imprinted genes, identify new imprinted genes, define imprinting regulation mechanisms, and study imprinting functions. [ABSTRACT FROM AUTHOR]

The 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. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. 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 compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues. [ABSTRACT FROM AUTHOR]

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development. [ABSTRACT FROM AUTHOR]

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32’s role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism. [ABSTRACT FROM AUTHOR]

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. Here, we show that Nos represses somatic gene expression by inhibiting translation of maternal importin-α2 (impα2) mRNA. Mis-expression of Impα2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, we propose that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a ‘double-lock’ mechanism to inhibit somatic gene expression in germline progenitors. [ABSTRACT FROM AUTHOR]

As small regulatory transcripts, microRNAs (miRs) act as genetic ‘fine tuners’ of posttranscriptional events, and as genetic switches to promote phenotypic switching. The miR miR26a targets the BMP signalling effector, smad1. We show that loss of miR26a leads to hemorrhage (a loss of vascular stability) in vivo, suggesting altered vascular differentiation. Reduction in miR26a levels increases smad1 mRNA and phospho-Smad1 (pSmad1) levels. We show that increasing BMP signalling by overexpression of smad1 also leads to hemorrhage. Normalization of Smad1 levels through double knockdown of miR26a and smad1 rescues hemorrhage, suggesting a direct relationship between miR26a, smad1 and vascular stability. Using an in vivo BMP genetic reporter and pSmad1 staining, we show that the effect of miR26a on smooth muscle differentiation is non-autonomous; BMP signalling is active in embryonic endothelial cells, but not in smooth muscle cells. Nonetheless, increased BMP signalling due to loss of miR26a results in an increase in acta2-expressing smooth muscle cell numbers and promotes a differentiated smooth muscle morphology. Similarly, forced expression of smad1 in endothelial cells leads to an increase in smooth muscle cell number and coverage. Furthermore, smooth muscle phenotypes caused by inhibition of the BMP pathway are rescued by loss of miR26a. Taken together, our data suggest that miR26a modulates BMP signalling in endothelial cells and indirectly promotes a differentiated smooth muscle phenotype. Our data highlights how crosstalk from BMP-responsive endothelium to smooth muscle is important for smooth muscle differentiation. [ABSTRACT FROM AUTHOR]

Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular CMs and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from Hdac1 and Cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation in crg mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors. [ABSTRACT FROM AUTHOR]

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, and that otic patterning defects in hmx3a-/- mutants are a close phenocopy to those seen in fgf3-/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full 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. [ABSTRACT FROM AUTHOR]

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development. [ABSTRACT FROM AUTHOR]

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition. [ABSTRACT FROM AUTHOR]

Rare individuals with inactivating mutations in the Huntington's disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development. [ABSTRACT FROM AUTHOR]

The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study—Iyer and colleagues []—defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions. [ABSTRACT FROM AUTHOR]

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated “mutation negative” probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA–and by implication rI production—correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA. [ABSTRACT FROM AUTHOR]

Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, parade, exhibit ectopic pigment cells localised to the ventral trunk, but also supernumerary cells restricted to the Ventral Stripe. Contrary to expectations, these melanocytes and iridophores are discrete cells, but closely apposed. We show that parade encodes Endothelin receptor Aa, expressed in the blood vessels, most prominently in the medial blood vessels, consistent with the ventral trunk phenotype. We provide evidence that neuronal fates are not affected in parade mutants, arguing against transdifferentiation of sympathetic neurons to pigment cells. We show that inhibition of BMP signaling prevents specification of sympathetic neurons, indicating conservation of this molecular mechanism with chick and mouse. However, inhibition of sympathetic neuron differentiation does not enhance the parade phenotype. Instead, we pinpoint ventral trunk-restricted proliferation of neural crest cells as an early feature of the parade phenotype. Importantly, using a chemical genetic screen for rescue of the ectopic pigment cell phenotype of parade mutants (whilst leaving the embryonic pattern untouched), we identify ErbB inhibitors as a key hit. The time-window of sensitivity to these inhibitors mirrors precisely the window defined previously as crucial for the setting aside of APSCs in the embryo, strongly implicating adult pigment stem cells as the source of the ectopic pigment cells. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels. [ABSTRACT FROM AUTHOR]

Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of nkx2.3+ pouch progenitors in zebrafish embryos and demonstrated an essential requirement of ectodermal BMP2b for their specification.At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgfαa rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification. [ABSTRACT FROM AUTHOR]

Endogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch of the 26G-RNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we explore other facets of 26G-RNA biology related to the ALG-3/4 branch. We find that sRNA abundance, sRNA pattern of origin and the 3’ UTR length of target transcripts are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we provide evidence suggesting that ALG-3 and ALG-4 regulate their own mRNAs in a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans. [ABSTRACT FROM AUTHOR]

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans. [ABSTRACT FROM AUTHOR]

Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP’s role in organ development and disorders associated with TCTP miss-expression. [ABSTRACT FROM AUTHOR]

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb. [ABSTRACT FROM AUTHOR]

The mechanisms that ensure fertilization of egg by a sperm are not fully understood. In all teleosts, a channel called the ‘micropyle’ is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the vitelline envelope by a single specialized follicle cell, the micropylar cell. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish (Danio rerio). Genome editing mutants affecting taz can grow to adults. However, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. We find that taz mutant eggs have no micropyle. Taz protein is specifically enriched in mid-oogenesis in the micropylar cell located at the animal pole of wild type oocyte, where it might regulate the cytoskeleton. Taz protein and micropylar cells are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. Here, we show by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Furthermore, we demonstrate that Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. Our data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade. [ABSTRACT FROM AUTHOR]

Identification of the potential donor(s) of human germline-derived cells is an issue in many criminal investigations and in paternity testing. The experimental and statistical methodology necessary to work up such cases is well established but may be more challenging if monozygotic (MZ) twins are involved. Then, elaborate genome-wide searches are required for the detection of early somatic mutations that distinguish the cell sample and its donor from the other twin, usually relying upon reference material other than semen (e.g. saliva). The first such cases, involving either criminal sexual offenses or paternity disputes, have been processed successfully by Eurofins Genomics and Forensics Campus. However, when presenting the experimental results in court, common forensic genetic practice requires that the residual uncertainty about donorship is quantified in the form of a likelihood ratio (LR). Hence, we developed a general mathematical framework for LR calculation, presented herein, which allows quantification of the evidence in favour of the true donor in the respective cases, based upon observed DNA sequencing read counts. [ABSTRACT FROM AUTHOR]

The earliest stage of animal development is controlled by maternally deposited mRNA transcripts and proteins. Once the zygote is able to transcribe its own genome, maternal transcripts are degraded, in a tightly regulated process known as the maternal to zygotic transition (MZT). While this process has been well-studied within model species, we have little knowledge of how the pools of maternal and zygotic transcripts evolve. To characterize the evolutionary dynamics and functional constraints on early embryonic expression, we created a transcriptomic dataset for 14 Drosophila species spanning over 50 million years of evolution, at developmental stages before and after the MZT, and compared our results with a previously published Aedes aegypti developmental time course. We found deep conservation over 250 million years of a core set of genes transcribed only by the zygote. This select group is highly enriched in transcription factors that play critical roles in early development. However, we also identify a surprisingly high level of change in the transcripts represented at both stages over the phylogeny. While mRNA levels of genes with maternally deposited transcripts are more highly conserved than zygotic genes, those maternal transcripts that are completely degraded at the MZT vary dramatically between species. We also show that hundreds of genes have different isoform usage between the maternal and zygotic genomes. Our work suggests that maternal transcript deposition and early zygotic transcription are remarkably dynamic over evolutionary time, despite the widespread conservation of early developmental processes. [ABSTRACT FROM AUTHOR]

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS. [ABSTRACT FROM AUTHOR]

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway. [ABSTRACT FROM AUTHOR]