Andrea Pauli

Research Institute of Molecular Pathology | IMP · Pauli Group

Mitochondrial ATP production is essential for development, yet the mechanisms underlying the continuous increase in mitochondrial activity during embryogenesis remain elusive. Using zebrafish as a model system for vertebrate development, we comprehensively profile mitochondrial activity, morphology, metabolome, proteome and phospho-proteome as well...

A large number of mRNAs of maternal origin are produced during oogenesis and deposited in the oocyte. Since transcription stops at the onset of meiosis during oogenesis and does not resume until later in embryogenesis, maternal mRNAs are the only templates for protein synthesis during this period. To ensure that a protein is made in the right place...

tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether...

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA ta...

Methylation of cytosines in the CG context (mCG) is the most abundant DNA modification in vertebrates that plays crucial roles in cellular differentiation and identity. After fertilization, DNA methylation patterns inherited from parental gametes are remodelled into a state compatible with embryogenesis. In mammals, this is achieved through the glo...

Fertilization, the fusion of sperm and egg, is essential for sexual reproduction. While several proteins have been demonstrated to be essential for the binding and fusion of gametes in vertebrates, the molecular mechanisms driving this key process are poorly understood. Here, we performed a protein interaction screen using AlphaFold-Multimer to unc...

Molecular compatibility between gametes is a prerequisite for successful fertilization. As long as a sperm and egg can recognize and bind each other via their surface proteins, gamete fusion may occur even between members of separate species, resulting in hybrids that can impact speciation. The egg membrane protein Bouncer confers species specifici...

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA ta...

Methylation of cytosines in the CG context (mCG) is the most abundant DNA modification in vertebrates that plays crucial roles in cellular differentiation and identity. After fertilisation, DNA methylation patterns inherited from parental gametes are remodelled into a state compatible with embryogenesis. In mammals, this is achieved through the glo...

The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts pre...

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1–4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribo...

The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has condi...

The sculpting of germ layers during gastrulation relies on the coordinated migration of progenitor cells, yet the cues controlling these long-range directed movements remain largely unknown. While directional migration often relies on a chemokine gradient generated from a localized source, we find that zebrafish ventrolateral mesoderm is guided by...

All sexually reproducing organisms depend on fertilization to survive as species. Despite the importance of fertilization, the mechanisms that drive sperm-egg compatibility are poorly understood. In fish, the egg protein Bouncer is necessary for fertilization and is species-specific between medaka and zebrafish. Here, we investigate whether Bouncer...

The maternal-to-zygotic transition (MZT) is a key developmental process in metazoan embryos that involves the activation of zygotic transcription (ZGA) and degradation of maternal transcripts. We employed metabolic mRNA sequencing (SLAMseq) to deconvolute the compound embryonic transcriptome in zebrafish. While mitochondrial zygotic transcripts pre...

The process of sperm-egg fusion is critical for successful fertilization, yet the underlying mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. m...

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that o...

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expres...

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, y...

The sculpting of germ layers during gastrulation relies on coordinated migration of progenitor cells, yet the cues controlling these long-range directed movements remain largely unknown. While directional migration often relies on a chemokine gradient generated from a localized source, we find that zebrafish ventrolateral mesoderm is guided by the...

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, y...

After fertilization, the sperm and egg contribute unequally to the newly formed zygote. While the sperm contributes mainly paternal DNA, the egg provides both maternal DNA and the bulk of the future embryonic cytoplasm. Most embryonic processes (like the onset of zygotic transcription) depend on maternally-provided cytoplasmic components, and it is...

Transposable elements threaten genome stability, and the Piwi-piRNA system has evolved to silence transposons in the germline. However, it remains largely unknown what mechanisms are utilized in early vertebrate embryos prior to germline establishment and ping-pong piRNA production. To address this, we first characterized small RNAs in early zebraf...

Ribosomes are produced in large quantities during oogenesis and stored in the egg. However, the egg and early embryo are translationally repressed. Using mass-spectrometry and cryo-EM analyses of ribosomes isolated from zebrafish and Xenopus eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant to an active state...

Significance We show that Bouncer’s homolog in mammals, SPACA4, is required for efficient fertilization in mice. In contrast to fish, in which Bouncer is required for female fertility, SPACA4 is expressed exclusively in the sperm and is required for male fertility. SPACA4 and Bouncer present an intriguing example of homologous proteins that both pl...

Fertilization is a multistep process that culminates in the fusion of sperm and egg, thus marking the beginning of a new organism in sexually reproducing species. Despite its importance for reproduction, the molecular mechanisms that regulate this singular event, particularly sperm-egg fusion, have remained mysterious for many decades. Here, we sum...

The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables rapid generation of conditional alleles via one-step zygote injection. SCON has conditional intronic function in various vertebrate species and its target insertion is as simple as CRISPR/Cas9...

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious...

Alternative cleavage and polyadenylation generates mRNA 3′ isoforms in a cell type-specific manner. Due to finite available RNA sequencing data of organisms with vast cell type complexity, currently available gene annotation resources are incomplete, which poses significant challenges to the comprehensive interpretation and quantification of transc...

The process of sperm-egg fusion is critical for successful fertilization, yet the underpinning mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D....

Self-organization is a key feature of many biological and developmental processes, including cell migration. Although cell migration has traditionally been viewed as a biological response to extrinsic signals, advances within the past two decades have highlighted the importance of intrinsic self-organizing properties to direct cell migration on mul...

Post-transcriptional regulation of gene expression is crucial during the oocyte-to-embryo transition, a highly dynamic process characterized by the absence of nuclear transcription. Thus, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified,...

Viral pandemics, such as Covid-19, pose serious threats to human societies. To control the spread of highly contagious viruses such as SARS-CoV-2, effective test-trace-isolate strategies require population-wide, systematic testing. Currently, RT-qPCR on extracted RNA is the only broadly accepted test for SARS-CoV-2 diagnostics, which bears the risk...

Cell fate transitions depend on balanced rewiring of transcription and translation programmes to mediate ordered developmental progression. Here we identify a feedback loop between nonsense-mediated mRNA decay (NMD) and translation initiation. We show that NMD controls the translation initiation factor Eif4a2 and its premature termination codon enc...

Global efforts to monitor and contain the Covid-19 pandemic, caused by the beta-coronavirus SARS-CoV-2, currently rely on RT-qPCR-based diagnostic assays. Yet their high cost, moderate throughput, and dependence on sophisticated equipment limit a broad implementation. Loop-mediated isothermal amplification (RT-LAMP) is an alternative detection meth...

Post-transcriptional mechanisms are crucial for the regulation of gene expression. These mechanisms are particularly important during rapid developmental transitions such as the oocyte-to-embryo transition, which is characterized by dramatic changes to the developmental program in the absence of nuclear transcription. Under these conditions, change...

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for...

The perpetuation and preservation of distinct species rely on mechanisms that ensure that only interactions between gametes of the same species can give rise to viable and fertile offspring. Species-specificity can act at various stages, ranging from physical/behavioral pre-copulatory mechanisms, to pre-zygotic incompatibility during fertilization,...

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for...

Bouncer keeps fertilization specific Fertilization needs to be highly efficient while remaining species-specific. However, despite decades of research, it is still unclear how these two requirements are met. Herberg et al. report the discovery of the Ly6/uPAR-type protein Bouncer as a species-specific fertilization factor in zebrafish (see the Pers...

Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. Here, we identify an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, as a crucial fertilization factor in zebrafish. We show that membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Re...

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhi...

MATLAB image processing scripts.

Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhi...

Toddler/Apela/Elabela is a conserved secreted peptide that regulates mesendoderm development during zebrafish gastrulation. Two non-exclusive models have been proposed to explain Toddler function. The ‘specification model’ postulates that Toddler signaling enhances Nodal signaling to properly specify endoderm, whereas the ‘migration model’ posits t...

Gene information for variable genes in RNA-Seq data set. Analysis with Cufflinks identified 47 genes with significantly different expression at one or more stages in the time course. Spreadsheet contains gene information and FPKM expression values for the variable genes in wild-type and toddler mutant embryos for each stage.

Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela re...

Translation is best known as the fundamental mechanism by which the ribosome converts a sequence of nucleotides into a string of amino acids. Extensive research over many years has elucidated the key principles of translation, and the majority of translated regions were thought to be known. The recent discovery of wide-spread translation outside of...

Upstream open reading frames (uORFs) are ubiquitous repressive genetic elements in vertebrate mRNAs. While much is known about the regulation of individual genes by their uORFs, the range of uORF-mediated translational repression in vertebrate genomes is largely unexplored. Moreover, it is unclear whether the repressive effects of uORFs are conserv...

Supplementary Figures 1-14, Supplementary Tables 1-7 and Supplementary Note 1

Conversions of iPython/Jupyter notebooks documenting all analysis for manuscript. Latest versions of iPython/Jupyer notebooks are available at https://github.com/chewgl/uORF_repressiveness_supplemental

Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compar...

Over the past decade, high-throughput studies have identified many novel transcripts. While their existence is undisputed, their coding potential and functionality have remained controversial. Recent computational approaches guided by ribosome profiling have indicated that translation is far more pervasive than anticipated and takes place on many t...

Cells control dynamic transitions in transcript levels by regulating transcription, processing, and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level; editing sites; and transcription, processing, and degradation...

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesi...

It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mes...

The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of...

Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput...

Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To id...

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their func...

Non-coding RNAs (ncRNAs) are emerging as key regulators of embryogenesis. They control embryonic gene expression by several means, ranging from microRNA-induced degradation of mRNAs to long ncRNA-mediated modification of chromatin. Many aspects of embryogenesis seem to be controlled by ncRNAs, including the maternal-zygotic transition, the maintena...

Developmental abnormalities observed in Cornelia de Lange syndrome have been genetically linked to mutations in the cohesin machinery. These and other recent experimental findings have led to the suggestion that cohesin, in addition to its canonical function of mediating sister chromatid cohesion, might also be involved in regulating gene expressio...

The metaphase-anaphase transition is orchestrated through proteolysis of numerous proteins by a ubiquitin protein ligase called the anaphase-promoting complex or cyclosome (APC/C). A crucial aspect of this process is sister chromatid separation, which is thought to be mediated by separase, a thiol protease activated by the APC/C. Separase cleaves c...

Cohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the alpha kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postm...

Progression into mitosis 14 after Rad21TEV cleavage by zygotic UAS-TEV expression. Time-lapse in vivo imaging of an embryo expressing His2Av-mRFP1 and EGFP-Cid was performed. Technical details were as described for Supplemental Movie 1. Two cells enter mitosis soon after the start of the movie followed by premature separation of sister chromatids.

Progression through mitosis 14 of Drosophila embryogenesis is illustrated as observed by time-lapse in vivo imaging of a control embryo expressing His2Av-mRFP1 and EGFP-Cid, but not UAS-TEV. A maximum projection of five confocal sections with 600 nm step size acquired at 15 second intervals using a 100× (NA 1.4) objective is presented. Several epid...

Movie showing the locomotion of a third-instar control larva (genotype: Cha-Gal4/+; Rad21ex3, Rad21/Rad21ex3, UAS-TEV) on nonnutritive agar.

Movie showing the locomotion of a third-instar larva after TEV cleavage of Rad21TEV in cholinergic neurons (genotype: Cha-Gal4/+; Rad21ex15, Rad21TEV/Rad21ex3, UAS-TEV).

Adaptation and exit from the mitotic arrest resulting from cleavage of Rad21TEV by zygotic UAS-TEV expression. Time-lapse in vivo imaging of an embryo expressing His2Av-mRFP1 and EGFP-Cid was performed. A maximum projection of five confocal sections with 600 nm step size acquired at 15 second intervals using a 40× (NA 1.25) objective is presented....

Chromatid behavior during the mitotic arrest in cycle 14 after cleavage of Rad21TEV by zygotic UAS-TEV expression. Time-lapse in vivo imaging of an embryo expressing His2Av-mRFP1 and EGFP-Cid was performed. Five confocal sections with 600 nm step size were acquired at 15 second intervals using a 100× (NA 1.4) objective. Single sections are shown in...

Ribosomal precursor particles are initially assembled in the nucleolus prior to their transfer to the nucleoplasm and export to the cytoplasm. In a screen to identify thermosensitive (ts) mutants defective in the export of pre-60S ribosomal subunit, we isolated the rix16-1 mutant. In this strain, nucleolar accumulation of the Rpl25-eGFP reporter wa...