Author Correction: The mid-developmental transition and the evolution of animal body plans.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Engineered heart tissue models from hiPSC-derived cardiomyocytes and cardiac ECM for disease modeling and drug testing applications.

Cardiac tissue engineering provides unique opportunities for cardiovascular disease modeling, drug testing, and regenerative medicine applications. To recapitulate human heart tissue, we combined human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a chitosan-enhanced extracellular-matrix (ECM) hydrogel, derived from decellularized pig hearts. Ultrastructural characterization of the ECM-derived engineered heart tissues (ECM-EHTs) revealed an anisotropic muscle structure, with embedded cardiomyocytes showing more mature properties than 2D-cultured hiPSC-CMs. Force measurements confirmed typical force-length relationships, sensitivity to extracellular calcium, and adequate ionotropic responses to contractility modulators. By combining genetically-encoded calcium and voltage indicators with laser-confocal microscopy and optical mapping, the electrophysiological and calcium-handling properties of the ECM-EHTs could be studied at the cellular and tissue resolutions. This allowed to detect drug-induced changes in contraction rate (isoproterenol, carbamylcholine), optical signal morphology (E-4031, ATX2, isoproterenol, ouabin and quinidine), cellular arrhythmogenicity (E-4031 and ouabin) and alterations in tissue conduction properties (lidocaine, carbenoxolone and quinidine). Similar assays in ECM-EHTs derived from patient-specific hiPSC-CMs recapitulated the abnormal phenotype of the long QT syndrome and catecholaminergic polymorphic ventricular tachycardia. Finally, programmed electrical stimulation and drug-induced pro-arrhythmia led to the development of reentrant arrhythmias in the ECM-EHTs. In conclusion, a novel ECM-EHT model was established, which can be subjected to high-resolution long-term serial functional phenotyping, with important implications for cardiac disease modeling, drug testing and precision medicine. STATEMENT OF SIGNIFICANCE: One of the main objectives of cardiac tissue engineering is to create an in-vitro muscle tissue surrogate of human heart tissue. To this end, we combined a chitosan-enforced cardiac-specific ECM hydrogel derived from decellularized pig hearts with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from healthy-controls and patients with inherited cardiac disorders. We then utilized genetically-encoded calcium and voltage fluorescent indicators coupled with unique optical imaging techniques and force-measurements to study the functional properties of the generated engineered heart tissues (EHTs). These studies demonstrate the unique potential of the new model for physiological and pathophysiological studies (assessing contractility, conduction and reentrant arrhythmias), novel disease modeling strategies ("disease-in-a-dish" approach) for studying inherited arrhythmogenic disorders, and for drug testing applications (safety pharmacology).

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells.

Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron's properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation.

The mid-developmental transition and the evolution of animal body plans.

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.