An EvoDevo Study of Salmonid Visual Opsin Dynamics and Photopigment Spectral Sensitivity

Salmonids are ideal models as many species follow a distinct developmental program from demersal eggs and a large yolk sac to hatching at an advanced developmental stage. Further, these economically important teleosts inhabit both marine- and freshwaters and experience diverse light environments during their life histories. At a genome level, salmonids have undergone a salmonid-specific fourth whole genome duplication event (Ss4R) compared to other teleosts that are already more genetically diverse compared to many non-teleost vertebrates. Thus, salmonids display phenotypically plastic visual systems that appear to be closely related to their anadromous migration patterns. This is most likely due to a complex interplay between their larger, more gene-rich genomes and broad spectrally enriched habitats; however, the molecular basis and functional consequences for such diversity is not fully understood. This study used advances in genome sequencing to identify the repertoire and genome organization of visual opsin genes (those primarily expressed in retinal photoreceptors) from six different salmonids [Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Chinook salmon (Oncorhynchus tshawytcha), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka)] compared to the northern pike (Esox lucius), a closely related non-salmonid species. Results identified multiple orthologues for all five visual opsin classes, except for presence of a single short-wavelength-sensitive-2 opsin gene. Several visual opsin genes were not retained after the Ss4R duplication event, which is consistent with the concept of salmonid rediploidization. Developmentally, transcriptomic analyzes of Atlantic salmon revealed differential expression within each opsin class, with two of the long-wavelength-sensitive opsins not being expressed before first feeding. Also, early opsin expression in the retina was located centrally, expanding dorsally and ventrally as eye development progressed, with rod opsin being the dominant visual opsin post-hatching. Modeling by spectral tuning analysis and atomistic molecular simulation, predicted the greatest variation in the spectral peak of absorbance to be within the Rh2 class, with a ∼40 nm difference in λmax values between the four medium-wavelength-sensitive photopigments. Overall, it appears that opsin duplication and expression, and their respective spectral tuning profiles, evolved to maximize specialist color vision throughout an anadromous lifecycle, with some visual opsin genes being lost to tailor marine-based vision.publishedVersio

Defining the role of Class II Phosphoinositide 3-Kinases in platelet function.

Introduction: Arterial thrombosis causes heart attack and ischaemic stroke and is the leading cause of death in the Western world. Platelets are the key blood cell responsible for the development of arterial thrombosis and understanding signalling pathways which lead to the incorporation of platelets into these thrombi may lead to the development of new and improved anti-thrombotic therapies. The phosphoinositide 3-kinases (PI3Ks) are a family of 3 classes (I, II, and III) of intracellular enzymes which generate lipid second messengers important for many cell signalling events. The role of Class I PI3Ks in platelets has been well defined and drugs which block Class I PI3K function are in pre-clinical trials as anti-thrombotics. However, virtually nothing is known about the Class II PI3Ks in platelets. Aim: The studies of this thesis used genetic mouse models to examine the role of the Class II PI3Ks in platelets. Key Findings: Mouse platelets express two of the three Class II PI3K isoforms, PI3K-C2 alpha and PI3K-C2 beta. The function of platelets isolated from PI3K-C2 beta-/- mice was largely normal. PI3K-C2alpha-/- mice were generated but died in utero prior to haematopoietic cell development, and were affected as early as 7.5 days post-conception. However, PI3K-C2 alpha+/- mice exhibited platelet-dependent defects in haemostasis and thrombosis. Specifically, anticoagulated PI3K-C2 alpha+/- mice had a 3-fold increase in tail bleeding time over littermate wild-type mice – an impairment comparable to that observed in anticoagulated wild-type mice treated with therapeutic doses of the gold standard anti-platelet agent, aspirin. Bone marrow reconstitution studies confirmed that the impaired in vivo haemostasis of PI3K-C2 alpha+/- mice was dependent on haematopoietic cells. An ex vivo whole blood thrombosis assay revealed PI3K-C2 alpha+/- mice formed thrombi that were larger (3-fold increase) but more unstable (embolization rate of 75% versus 0%) than those of littermate wild-type mice. Detailed analyses of the function of platelets from PI3K-C2 alpha+/- mice revealed a decrease in adhesion via the collagen receptor(s), GPVI and/or alpha2beta1, and a shear-dependent increase in platelet adhesion via the integrin alphaIIbbeta3 – a phenotype which was reproduced in wild-type platelets treated with alcohols to pharmacologically increase plasma membrane fluidity. Finally, a novel mouse genetic model was developed to further reduce PI3K-C2alpha expression levels in platelets using an inducible and reversible RNAi-based approach. Platelets from these PI3K-C2 alpha-deficient mice expressed < 10% of normal PI3K-C2 alpha levels and these mice exhibited a significantly more severe in vivo haemostatic defect than that observed in PI3K-C2 alpha+/- mice. Conclusions: The studies of this thesis define a novel role for the Class II PI3Ks in platelet biology. Examination of platelet function in mice genetically deficient in the Class II PI3Ks using a combination of in vitro platelet function studies, ex vivo thrombosis assays, and in vivo models, demonstrated that PI3K-C2 alpha regulates the function of cell surface platelet adhesion receptors, possibly through changes in plasma membrane structure and/or function. These studies suggest that further investigation of the function of PI3K-C2 alpha in platelet biology is warranted to determine whether this lipid kinase represents a novel target for the development of anti-platelet agents for the prevention of arterial thrombosis

Defining the role of Class II Phosphoinositide 3-Kinases in platelet function.

Introduction: Arterial thrombosis causes heart attack and ischaemic stroke and is the leading cause of death in the Western world. Platelets are the key blood cell responsible for the development of arterial thrombosis and understanding signalling pathways which lead to the incorporation of platelets into these thrombi may lead to the development of new and improved anti-thrombotic therapies. The phosphoinositide 3-kinases (PI3Ks) are a family of 3 classes (I, II, and III) of intracellular enzymes which generate lipid second messengers important for many cell signalling events. The role of Class I PI3Ks in platelets has been well defined and drugs which block Class I PI3K function are in pre-clinical trials as anti-thrombotics. However, virtually nothing is known about the Class II PI3Ks in platelets. Aim: The studies of this thesis used genetic mouse models to examine the role of the Class II PI3Ks in platelets. Key Findings: Mouse platelets express two of the three Class II PI3K isoforms, PI3K-C2 alpha and PI3K-C2 beta. The function of platelets isolated from PI3K-C2 beta-/- mice was largely normal. PI3K-C2alpha-/- mice were generated but died in utero prior to haematopoietic cell development, and were affected as early as 7.5 days post-conception. However, PI3K-C2 alpha+/- mice exhibited platelet-dependent defects in haemostasis and thrombosis. Specifically, anticoagulated PI3K-C2 alpha+/- mice had a 3-fold increase in tail bleeding time over littermate wild-type mice – an impairment comparable to that observed in anticoagulated wild-type mice treated with therapeutic doses of the gold standard anti-platelet agent, aspirin. Bone marrow reconstitution studies confirmed that the impaired in vivo haemostasis of PI3K-C2 alpha+/- mice was dependent on haematopoietic cells. An ex vivo whole blood thrombosis assay revealed PI3K-C2 alpha+/- mice formed thrombi that were larger (3-fold increase) but more unstable (embolization rate of 75% versus 0%) than those of littermate wild-type mice. Detailed analyses of the function of platelets from PI3K-C2 alpha+/- mice revealed a decrease in adhesion via the collagen receptor(s), GPVI and/or alpha2beta1, and a shear-dependent increase in platelet adhesion via the integrin alphaIIbbeta3 – a phenotype which was reproduced in wild-type platelets treated with alcohols to pharmacologically increase plasma membrane fluidity. Finally, a novel mouse genetic model was developed to further reduce PI3K-C2alpha expression levels in platelets using an inducible and reversible RNAi-based approach. Platelets from these PI3K-C2 alpha-deficient mice expressed < 10% of normal PI3K-C2 alpha levels and these mice exhibited a significantly more severe in vivo haemostatic defect than that observed in PI3K-C2 alpha+/- mice. Conclusions: The studies of this thesis define a novel role for the Class II PI3Ks in platelet biology. Examination of platelet function in mice genetically deficient in the Class II PI3Ks using a combination of in vitro platelet function studies, ex vivo thrombosis assays, and in vivo models, demonstrated that PI3K-C2 alpha regulates the function of cell surface platelet adhesion receptors, possibly through changes in plasma membrane structure and/or function. These studies suggest that further investigation of the function of PI3K-C2 alpha in platelet biology is warranted to determine whether this lipid kinase represents a novel target for the development of anti-platelet agents for the prevention of arterial thrombosis

An EvoDevo Study of Salmonid Visual Opsin Dynamics and Photopigment Spectral Sensitivity

Salmonids are ideal models as many species follow a distinct developmental program from demersal eggs and a large yolk sac to hatching at an advanced developmental stage. Further, these economically important teleosts inhabit both marine- and freshwaters and experience diverse light environments during their life histories. At a genome level, salmonids have undergone a salmonid-specific fourth whole genome duplication event (Ss4R) compared to other teleosts that are already more genetically diverse compared to many non-teleost vertebrates. Thus, salmonids display phenotypically plastic visual systems that appear to be closely related to their anadromous migration patterns. This is most likely due to a complex interplay between their larger, more gene-rich genomes and broad spectrally enriched habitats; however, the molecular basis and functional consequences for such diversity is not fully understood. This study used advances in genome sequencing to identify the repertoire and genome organization of visual opsin genes (those primarily expressed in retinal photoreceptors) from six different salmonids [Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Chinook salmon (Oncorhynchus tshawytcha), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka)] compared to the northern pike (Esox lucius), a closely related non-salmonid species. Results identified multiple orthologues for all five visual opsin classes, except for presence of a single short-wavelength-sensitive-2 opsin gene. Several visual opsin genes were not retained after the Ss4R duplication event, which is consistent with the concept of salmonid rediploidization. Developmentally, transcriptomic analyzes of Atlantic salmon revealed differential expression within each opsin class, with two of the long-wavelength-sensitive opsins not being expressed before first feeding. Also, early opsin expression in the retina was located centrally, expanding dorsally and ventrally as eye development progressed, with rod opsin being the dominant visual opsin post-hatching. Modeling by spectral tuning analysis and atomistic molecular simulation, predicted the greatest variation in the spectral peak of absorbance to be within the Rh2 class, with a ∼40 nm difference in λmax values between the four medium-wavelength-sensitive photopigments. Overall, it appears that opsin duplication and expression, and their respective spectral tuning profiles, evolved to maximize specialist color vision throughout an anadromous lifecycle, with some visual opsin genes being lost to tailor marine-based vision