iPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the gene (p.

Chromosome segregation fidelity requires microtubule polyglutamylation by the cancer downregulated enzyme TTLL11.

Regulation of microtubule (MT) dynamics is key for mitotic spindle assembly and faithful chromosome segregation. Here we show that polyglutamylation, a still understudied post-translational modification of spindle MTs, is essential to define their dynamics within the range required for error-free chromosome segregation. We identify TTLL11 as an enzyme driving MT polyglutamylation in mitosis and show that reducing TTLL11 levels in human cells or zebrafish embryos compromises chromosome segregation fidelity and impairs early embryonic development.

Specialization of the photoreceptor transcriptome by -dependent microexons is required for outer segment maintenance and vision.

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport.

Cooperative epithelial phagocytosis enables error correction in the early embryo.

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown.

The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior.

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations.

GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation.

Engineered Macroscale Cardiac Constructs Elicit Human Myocardial Tissue-like Functionality.

In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Here, we report on the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSCs), which we term "CardioSlice.

Proteomics Analysis of Extracellular Matrix Remodeling During Zebrafish Heart Regeneration.

Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored.

CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons.

Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus.

Reprogramming Captures the Genetic and Tumorigenic Properties of Neurofibromatosis Type 1 Plexiform Neurofibromas.

Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development.

Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the "safe harbor" AAVS1 locus.

We report the generation-characterization of a fetal liver (FL) B-cell progenitor (BCP)-derived human induced pluripotent stem cell (hiPSC) line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the "safe locus" AAVS1 (iCas9-FL-BCP-hiPSC). Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot.

Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks.

Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration.

Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease.

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC.

Stepwise assembly of the Nova-regulated alternative splicing network in the vertebrate brain.

Novel organismal structures in metazoans are often undergirded by complex gene regulatory networks; as such, understanding the emergence of new structures through evolution requires reconstructing the series of evolutionary steps leading to these underlying networks. Here, we reconstruct the step-by-step assembly of the vertebrate splicing network regulated by Nova, a splicing factor that modulates alternative splicing in the vertebrate central nervous system by binding to clusters of YCAY motifs on pre-RNA transcripts. Transfection of human HEK293T cells with Nova orthologs indicated vertebrate-like splicing regulatory activity in bilaterian invertebrates, thus Nova acquired the ability to bind YCAY clusters and perform vertebrate-like splicing modulation at least before the last common ancestor of bilaterians.

Implications of duplicated cis-regulatory elements in the evolution of metazoans: the DDI model or how simplicity begets novelty.

The discovery that most regulatory genes were conserved among animals from distant phyla challenged the ideas that gene duplication and divergence of homologous coding sequences were the basis for major morphological changes in metazoan evolution. In recent years, however, the interest for the roles, conservation and changes of non-coding sequences grew-up in parallel with genome sequencing projects. Presently, many independent studies are highlighting the importance that subtle changes in cis-regulatory regions had in the evolution of morphology trough the Animal Kingdom.

From the American to the European amphioxus: towards experimental Evo-Devo at the origin of chordates.

Pallid anchovy fillet, friendly filtering, peacefully laying and little lancelet are some of the nicknames and adjectives the cephalochordate amphioxus has received throughout the last two centuries. Traditionally regarded as the living representative of the last ancestor of vertebrates, amphioxus has recently been promoted to the privileged position of being the most ancient chordate. The preliminary analysis of its prototypical genome is nearly completed, and its hidden secrets towards the understanding of the primitive chordate and deuterostome genomes will soon see the light.

Gene expansion and retention leads to a diverse tyrosine kinase superfamily in amphioxus.

Tyrosine kinase (TK) proteins play a central role in cellular behavior and development of animals. The expansion of this superfamily is regarded as a key event in the evolution of the complex signaling pathways and gene networks of metazoans and is a prominent example of how shuffling of protein modules may generate molecular novelties. Using the intron/exon structure within the TK domain (TK intron code) as a complementary tool for the assignment of orthology and paralogy, we identified and studied the 118 TK proteins of the amphioxus Branchiostoma floridae genome to elucidate TK gene family evolution in metazoans and chordates in particular.

Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum).

The cephalochordate amphioxus (Branchiostoma sp.) is an important animal model for studying the evolution of chordate developmental mechanisms. Obtaining amphioxus embryos is a key step for these studies.

Evolutionary genomics of the recently duplicated amphioxus Hairy genes.

Amphioxus Hairy genes have gone through a number of lineage-specific duplications, resulting in eight members, some of which are differentially expressed in the embryo. In order to gain insights into the evolution and function of this gene family we have compared their genomic structure and searched for conserved non-coding sequence elements. We have found that introns have been lost independently from these genes at least twice and after the duplication events.

The amphioxus Hairy family: differential fate after duplication.

Vertebrate Hairy genes are highly pleiotropic and have been implicated in numerous functions, such as somitogenesis, neurogenesis and endocrine tissue development. In order to gain insight into the timing of acquisition of these roles by the Hairy subfamily, we have cloned and studied the expression pattern of the Hairy gene(s) in amphioxus. The cephalochordate amphioxus is widely believed to be the living invertebrate more closely related to vertebrates, the genome of which has not undergone the massive gene duplications that took place early during vertebrate evolution.