Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart.

Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity in vitro, the in vivo functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, αBa and αBb We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in αBa than αBb mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.

Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.

The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were γB- and γD-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone α-crystallin in vitro To better understand the mechanism of the cataract phenotype, we transgenically expressed different γD-crystallin mutants in the zebrafish lens and observed a range of lens defects that arise primarily from the aggregation of the mutant proteins. Unlike mouse models, a strong correlation was observed between the severity and penetrance of the phenotype and the level of destabilization of the mutant. We interpret this result to reflect the presence of a proteostasis network that can "sense" protein stability. In the more destabilized mutants, the capacity of this network is overwhelmed, leading to the observed increase in phenotypic penetrance. Overexpression of αA-crystallin had no significant effects on the penetrance of lens defects, suggesting that its chaperone capacity is not limiting. Although consistent with the prevailing hypothesis that a chaperone network is required for lens transparency, our results suggest that αA-crystallin may not be efficient to inhibit aggregation of lens γ-crystallin. Furthermore, our work implicates additional inputs/factors in this underlying proteostasis network and demonstrates the utility of zebrafish as a platform to delineate mechanisms of cataract.

Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.

An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.

Redox-mediated quorum sensing in plants.

The rhizosphere, the narrow zone of soil around plant roots, is a complex network of interactions between plants, bacteria, and a variety of other organisms. The absolute dependence on host-derived signals, or xenognosins, to regulate critical developmental checkpoints for host commitment in the obligate parasitic plants provides a window into the rhizosphere's chemical dynamics. These sessile intruders use H2O2 in a process known as semagenesis to chemically modify the mature root surfaces of proximal host plants and generate p-benzoquinones (BQs). The resulting redox-active signaling network regulates the spatial and temporal commitments necessary for host attachment. Recent evidence from non-parasites, including Arabidopsis thaliana, establishes that reactive oxygen species (ROS) production regulates similar redox circuits related to root recognition, broadening xenognosins' role beyond the parasites. Here we compare responses to the xenognosin dimethoxybenzoquinone (DMBQ) between the parasitic plant Striga asiatica and the non-parasitic A. thaliana. Exposure to DMBQ simulates the proximity of a mature root surface, stimulating an increase in cytoplasmic Ca2+ concentration in both plants, but leads to remarkably different phenotypic responses in the parasite and non-parasite. In S. asiatica, DMBQ induces development of the host attachment organ, the haustorium, and decreases ROS production at the root tip, while in A. thaliana, ROS production increases and further growth of the root tip is arrested. Obstruction of Ca2+ channels and the addition of antioxidants both lead to a decrease in the DMBQ response in both parasitic and non-parasitic plants. These results are consistent with Ca2+ regulating the activity of NADPH oxidases, which in turn sustain the autocatalytic production of ROS via an external quinone/hydroquinone redox cycle. Mechanistically, this chemistry is similar to black and white photography with the emerging dynamic reaction-diffusion network laying the foundation for the precise temporal and spatial control underlying rhizosphere architecture.

Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors.

Malaria parasites increase their host erythrocyte's permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against in vitro parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel's structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.