Quantitative intravital imaging in zebrafish reveals in vivo dynamics of physiological-stress-induced mitophagy.

Mitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive fluorescent probes, mito-Keima and mito-EGFP-mCherry, and used quantitative intravital imaging to illuminate mitophagy during physiological stresses, namely, embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress response dynamics, and time-lapse imaging revealed that mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor (bnip3) prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes [bnip3la (nix), fundc1, pink1 or prkn (Parkin)] had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.

Fetal alcohol spectrum disorder predisposes to metabolic abnormalities in adulthood.

Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.

Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood Flow.

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.

A quantitative single-cell assay for retrograde membrane traffic enables rapid detection of defects in cellular organization.

Retrograde membrane trafficking from plasma membrane to Golgi and endoplasmic reticulum typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport. The approach can be applied in high throughput to elucidate the underlying biology of membrane traffic and how endosomes adapt to the physiologic needs of different cell types and cell states.

There Is Something Fishy About Liver Cancer: Zebrafish Models of Hepatocellular Carcinoma.

The incidence of hepatocellular carcinoma (HCC) and the mortality resulting from HCC are both increasing. Most patients with HCC are diagnosed at advanced stages when curative treatments are impossible. Current drug therapy extends mean overall survival by only a short period of time. Genetic mutations associated with HCC vary widely. Therefore, transgenic and mutant animal models are needed to investigate the molecular effects of specific mutations, classify them as drivers or passengers, and develop targeted treatments. Cirrhosis, however, is the premalignant state common to 90% of HCC patients. Currently, no specific therapies are available to halt or reverse the progression of cirrhosis to HCC. Understanding the genetic drivers of HCC as well as the biochemical, mechanical, hormonal, and metabolic changes associated with cirrhosis could lead to novel treatments and cancer prevention strategies. Although additional therapies recently received Food and Drug Administration approval, significant clinical breakthroughs have not emerged since the introduction of the multikinase inhibitor sorafenib, necessitating alternate research strategies. Zebrafish (Danio rerio) are effective for disease modeling because of their high degree of gene and organ architecture conservation with human beings, ease of transgenesis and mutagenesis, high fecundity, and low housing cost. Here, we review zebrafish models of HCC and identify areas on which to focus future research efforts to maximize the advantages of the zebrafish model system.

Forces of Change: Mechanics Underlying Formation of Functional 3D Organ Buds.

3D organ buds that can recapitulate organ function have myriad applications for regenerative and personalized medicine. Here, Takebe et al. (2015) describe a generalized method for organ bud formation, demonstrating that mechanosensitive mesenchymal stem cells drive condensation of heterotypic cell mixtures to create buds from diverse organs.

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins.

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate.

Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification.

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (>75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.

Glycosaminoglycan-binding hydrogels enable mechanical control of human pluripotent stem cell self-renewal.

Reaping the promise of human embryonic stem (hES) cells hinges on effective defined culture conditions. Efforts to identify chemically defined environments for hES cell propagation would benefit from understanding the relevant functional properties of the substratum. Biological materials are often employed as substrata, but their complexity obscures a molecular level analysis of their relevant attributes. Because the properties of hydrogels can be tuned and altered systematically, these materials can reveal the impact of substratum features on cell fate decisions. By tailoring the peptide displayed to cells and the substrate mechanical properties, a hydrogel was generated that binds hES cell surface glycosaminoglycans (GAGs) and functions robustly in a defined culture medium to support long-term hES cell self-renewal. A key attribute of the successful GAG-binding hydrogels is their stiffness. Only stiff substrates maintain hES cell proliferation and pluripotency. These findings indicate that cells can respond to mechanical information transmitted via GAG engagement. Additionally, we found that the stiff matrices afforded activation of the paralogous proteins YAP/TAZ, which are transcriptional coactivators implicated in mechanosensing and hES cell pluripotency. These results indicate that the substratum mechanics can be tuned to activate specific pathways linked to pluripotency. Because several different hES and induced pluripotent stem cell lines respond similarly, we conclude that stiff substrata are more effective for the long-term propagation of human pluripotent stem cells.

Small-molecule-modified surfaces engage cells through the αvß3 integrin.

Integrins play myriad and vital roles in development and disease. They connect a cell with its surroundings and transmit chemical and mechanical signals across the plasma membrane to the cell's interior. Dissecting their roles in cell behavior is complicated by their overlapping ligand specificity and shared downstream signaling components. In principle, immobilized synthetic peptides can mimic extracellular matrix proteins by supporting integrin-mediated adhesion, but most short peptide sequences lack selectivity for one integrin over others. In contrast, synthetic integrin antagonists can be highly selective. We hypothesized that this selectivity could be exploited if antagonists, when immobilized, could support cellular adhesion and activate signaling by engaging specific cell-surface integrins. To investigate this possibility, we designed a bifunctional (RGD)-based peptidomimetic for surface presentation. Our conjugate combines a high affinity integrin ligand with a biotin moiety; the former engages the α(v)ß(3) integrin, and the latter allows for presentation on streptavidin-coated surfaces. Surfaces decorated with this ligand promote both cellular adhesion and integrin activation. Moreover, the selectivity of these surfaces for the α(v)ß(3) integrin can be exploited to capture a subset of cells from a mixed population. We anticipate that surfaces displaying highly selective small molecule ligands can reveal the contributions of specific integrin heterodimers to cell adhesion and signaling.

A defined glycosaminoglycan-binding substratum for human pluripotent stem cells.

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.