Induction of kidney-related gene programs through co-option of SALL1 in mole ovotestes.

Changes in gene expression represent an important source of phenotypic innovation. Yet how such changes emerge and impact the evolution of traits remains elusive. Here, we explore the molecular mechanisms associated with the development of masculinizing ovotestes in female moles. By performing integrative analyses of epigenetic and transcriptional data in mole and mouse, we identified the co-option of SALL1 expression in mole ovotestes formation. Chromosome conformation capture analyses highlight a striking conservation of the 3D organization at the SALL1 locus, but an evolutionary divergence of enhancer activity. Interspecies reporter assays support the capability of mole-specific enhancers to activate transcription in urogenital tissues. Through overexpression experiments in transgenic mice, we further demonstrate the capability of SALL1 to induce kidney-related gene programs, which are a signature of mole ovotestes. Our results highlight the co-option of gene expression, through changes in enhancer activity, as a plausible mechanism for the evolution of traits.

Coordination of non-professional efferocytosis and actomyosin contractility during epithelial tissue morphogenesis.

In developing embryos, specific cell populations are often removed to remodel tissue architecture for organogenesis. During urinary tract development, an epithelial duct called the common nephric duct (CND) gets shortened and eventually eliminated to remodel the entry point of the ureter into the bladder. Here we show that non-professional efferocytosis (the process in which epithelial cells engulf apoptotic bodies) is the main mechanism that contributes to CND shortening. Combining biological metrics and computational modeling, we show that efferocytosis with actomyosin contractility are essential factors that drive the CND shortening without compromising the ureter-bladder structural connection. The disruption of either apoptosis, non-professional efferocytosis, or actomyosin results in contractile tension reduction and deficient CND shortening. Actomyosin activity helps to maintain tissue architecture while non-professional efferocytosis removes cellular volume. Together our results demonstrate that non-professional efferocytosis with actomyosin contractility are important morphogenetic factors controlling CND morphogenesis.

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Building a stem cell-based primate uterus.

The uterus is the organ for embryo implantation and fetal development. Most current models of the uterus are centred around capturing its function during later stages of pregnancy to increase the survival in pre-term births. However, in vitro models focusing on the uterine tissue itself would allow modelling of pathologies including endometriosis and uterine cancers, and open new avenues to investigate embryo implantation and human development. Motivated by these key questions, we discuss how stem cell-based uteri may be engineered from constituent cell parts, either as advanced self-organising cultures, or by controlled assembly through microfluidic and print-based technologies.

Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells.

Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis.

The mole genome reveals regulatory rearrangements associated with adaptive intersexuality.

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

Comparison of long-term versus short-term effects of okadaic acid on the apoptotic status of human HepaRG cells.

The biotoxin okadaic acid (OA) is a lipophilic secondary metabolite of marine microalgae. Therefore, OA accumulates in the fatty tissue of various shellfish and may thus enter the food chain. The ingestion of OA via contaminated marine species can lead to the diarrhetic shellfish poisoning syndrome characterized by the occurrence of a series of acute gastrointestinal symptoms in humans. In addition, genotoxicity and tumor-promoting properties of OA might constitute a long-term threat to human health. In order to deepen our understanding of the molecular effects of OA, we compared long-term (14 d) and short-term (24 h and 48 h) apoptotic effects of the compound on human HepaRG hepatocarcinoma cells. Cells were treated either with single doses for 24 and 48 h, respectively, or seven times over a period of 14 d, so that the cumulated quantities of OA in the long-term approach were equal to the single doses upon short-term treatment. Both short-term treatment scenarios led to the induction of apoptosis. Specific caspase activation assays and transcriptional analysis of mRNAs encoding proteins involved in the regulation of apoptosis suggest that OA-induced apoptosis occurs presumably by activation of the intrinsic apoptotic pathway. In contrast, effects were much less pronounced in case of long-term treatment. This is possibly linked to cellular protective mechanisms against low amounts of toxins, e.g. transporter-mediated efflux. In conclusion, our results show a clear concentration- and time-dependency of OA-mediated apoptotic effects in HepaRG cells and contribute to the elucidation of molecular effects of OA.

Genome-wide identification of alternative splicing events that regulate protein transport across the secretory pathway.

Alternative splicing (AS) strongly increases proteome diversity and functionality in eukaryotic cells. Protein secretion is a tightly controlled process, especially when it occurs in a tissue-specific and differentiation-dependent manner. While previous work has focussed on transcriptional and post-translational regulatory mechanisms, the impact of AS on the secretory pathway remains largely unexplored. Here, we integrate results from a published screen for modulators of protein transport and RNA-Seq analyses to identify over 200 AS events as secretion regulators. We confirm that splicing events along all stages of the secretory pathway regulate the efficiency of membrane trafficking using morpholino and CRISPR/Cas9 experiments. We furthermore show that these events are highly tissue-specific and mediate an adaptation of the secretory pathway during T-cell activation and adipocyte differentiation. Our data substantially advance the understanding of AS functionality, add a new regulatory layer to a fundamental cell biological process and provide a resource of alternative isoforms that control the secretory pathway.

Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9.

In flowering plant plastids and mitochondria, multiple organellar RNA editing factor (MORF/RIP) proteins are required at most sites for efficient C to U RNA editing catalyzed by the RNA editosome. MORF proteins harbor a conserved stretch of residues (MORF-box), form homo- and heteromers and interact with selected PPR (pentatricopeptide repeat) proteins, which recognize each editing site. The molecular function of the MORF-box remains elusive since it shares no sequence similarity with known domains. We determined structures of the A. thaliana mitochondrial MORF1 and chloroplast MORF9 MORF-boxes which both adopt a novel globular fold (MORF domain). Our structures state a paradigmatic model for MORF domains and their specific dimerization via a hydrophobic interface. We cross-validate the interface by yeast two-hybrid studies and pulldown assays employing structure-based mutants. We find a structural similarity of the MORF domain to an N-terminal ferredoxin-like domain (NFLD), which confers RNA substrate positioning in bacterial 4-thio-uracil tRNA synthetases, implying direct RNA contacts of MORF proteins during RNA editing. With the MORF1 and MORF9 structures we elucidate a yet unknown fold, corroborate MORF interaction studies, validate the mechanism of MORF multimerization by structure-based mutants and pave the way towards a complete structural characterization of the plant RNA editosome.