Modelling human blastocysts by reprogramming fibroblasts into iBlastoids.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1-4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.

The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production.

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

3D-cardiomics: A spatial transcriptional atlas of the mammalian heart.

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/.

Splicing factors as regulators of miRNA biogenesis - links to human disease.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally by fine-tuning mRNA levels and translation during development and in adult tissues. miRNAs are transcribed as parts of longer precursors that undergo multiple processing steps before the mature miRNAs reach their target mRNAs in the cytoplasm. In addition to Drosha/DGCR8 and Dicer that are the essential components of the miRNA processing pathway, a range of other RNA binding proteins have recently been implicated in miRNA biogenesis. Among these, several well-known splicing factors have emerged as regulators of distinct miRNAs. In this review, we examine the mechanisms by which splicing factors regulate miRNA biogenesis. As both splicing factors and miRNAs play central roles in human disease biology we discuss implications of the links between splicing factors and miRNAs in human disease.

Apicobasal RNA asymmetries regulate cell fate in the early mouse embryo.

The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate.

TINC- A Method to Dissect Regulatory Complexes at Single-Locus Resolution- Reveals an Extensive Protein Complex at the Nanog Promoter.

Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.