Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.</p

Lgl2 Executes Its Function as a Tumor Suppressor by Regulating ErbB Signaling in the Zebrafish Epidermis

Changes in tissue homeostasis, acquisition of invasive cell characteristics, and tumor formation can often be linked to the loss of epithelial cell polarity. In carcinogenesis, the grade of neoplasia correlates with impaired cell polarity. In Drosophila, lethal giant larvae (lgl), discs large (dlg), and scribble, which are components of the epithelial apico-basal cell polarity machinery, act as tumor suppressors, and orthologs of this evolutionary conserved pathway are lost in human carcinoma with high frequency. However, a mechanistic link between neoplasia and vertebrate orthologs of these tumor-suppressor genes remains to be fully explored at the organismal level. Here, we show that the pen/lgl2 mutant phenotype shares two key cellular and molecular features of mammalian malignancy: cell autonomous epidermal neoplasia and epithelial-to-mesenchymal-transition (EMT) of basal epidermal cells including the differential expression of several regulators of EMT. Further, we found that epidermal neoplasia and EMT in pen/lgl2 mutant epidermal cells is promoted by ErbB signalling, a pathway of high significance in human carcinomas. Intriguingly, EMT in the pen/lgl2 mutant is facilitated specifically by ErbB2 mediated E-cadherin mislocalization and not via canonical snail–dependent down-regulation of E-cadherin expression. Our data reveal that pen/lgl2 functions as a tumor suppressor gene in vertebrates, establishing zebrafish pen/lgl2 mutants as a valuable cancer model

Translational profiling through biotinylation of tagged ribosomes in zebrafish

Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling

Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise.

Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle