A. E. O. Trezise

Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a

Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, SCN5A, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene, Scn5a, causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch clamp analyses of isolated ventricular myocytes from adult Scn5a+/− mice demonstrate a ≈50% reduction in sodium conductance. Scn5a+/− hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently diverse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell–specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V–X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro–oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Ancient colour vision: multiple opsin genes in the ancestral vertebrates.

Molecular investigation of the origin of colour vision has discovered five visual pigment (opsin) genes, all of which are expressed in an agnathan (jawless) fish, the lamprey Geotria australis. Lampreys are extant representatives of an ancient group of vertebrates whose origins are thought to date back to at least the early Cambrian, approximately 540 million years ago [1.]. Phylogenetic analysis has identified the visual pigment opsin genes of G. australis as orthologues of the major classes of vertebrate opsin genes. Therefore, multiple opsin genes must have originated very early in vertebrate evolution, prior to the separation of the jawed and jawless vertebrate lineages, and thereby provided the genetic basis for colour vision in all vertebrate species. The southern hemisphere lamprey Geotria australis (Figure 1A,B) possesses a predominantly cone-based visual system designed for photopic (bright light) vision [2. S.P. Collin, I.C. Potter and C.R. Braekevelt, The ocular morphology of the southern hemisphere lamprey Geotria australis Gray, with special reference to optical specializations and the characterisation and phylogeny of photoreceptor types. Brain Behav. Evol. 54 (1999), pp. 96–111.2. and 3.]. Previous work identified multiple cone types suggesting that the potential for colour vision may have been present in the earliest members of this group. In order to trace the molecular evolution and origins of vertebrate colour vision, we have examined the genetic complement of visual pigment opsins in G. australis.

The influence of ontogeny and light environment on the expression of visual pigment opsins in the retina of the black bream, Acanthopagrus butcheri

SUMMARY The correlation between ontogenetic changes in the spectral absorption characteristics of retinal photoreceptors and expression of visual pigment opsins was investigated in the black bream, Acanthopagrus butcheri. To establish whether the spectral qualities of environmental light affected the complement of visual pigments during ontogeny, comparisons were made between fishes reared in: (1) broad spectrum aquarium conditions; (2) short wavelength-reduced conditions similar to the natural environment; or (3) the natural environment (wild-caught). Microspectrophotometry was used to determine the wavelengths of spectral sensitivity of the photoreceptors at four developmental stages: larval, post-settlement, juvenile and adult. The molecular sequences of the rod (Rh1) and six cone (SWS1, SWS2A and B, Rh2Aα and β, and LWS) opsins were obtained and their expression levels in larval and adult stages examined using quantitative RT-PCR. The changes in spectral sensitivity of the cones were related to the differing levels of opsin expression during ontogeny. During the larval stage the predominantly expressed opsin classes were SWS1, SWS2B and Rh2Aα, contrasting with SWS2A, Rh2Aβ and LWS in the adult. An increased proportion of long wavelength-sensitive double cones was found in fishes reared in the short wavelength-reduced conditions and in wild-caught animals, indicating that the expression of cone opsin genes is also regulated by environmental light.

Functional characterization, tuning, and regulation of visual pigment gene expression in an anadromous lamprey

Lampreys are one of the two surviving groups of jawless vertebrates, whose ancestors arose more than 540 million years ago. Some species, such as Geotria australis, are anadromous, commencing life as ammo‐coetes in rivers, migrating downstream to the sea, and migrating back into rivers to spawn. Five photoreceptor types and five retinal cone opsin genes (LWS, SWS1, SWS2, RhA, and RhB) have previously been identified in G. australis. This implies that the ancestral vertebrates pos‐sessed photopic or cone‐based vision with the potential for pentachromacy. Changes in the morphology of pho‐toreceptors and their spectral sensitivity are encountered during differing aquatic phases of the lamprey lifecycle. To understand the molecular basis for these changes, we characterized the visual pigments and measured the relative levels of opsin expression over two lifecycle phases that are accompanied by contrasting ambient light environments. By expressing recombinant opsins in vitro, we show that SWS1, SWS2, RhA, and RhB visual pigments possess λmax values of 359, 439, 497, and 492 nm respectively. For the LWS visual pigment, we predict a λmax value of 560 nm based on key spectral tuning sites in other vertebrate LWS opsins. Quantitative reverse transcriptase‐polymerase chain reaction reveals that the retinal opsin genes of G. australis are differentially regulated such that the visual system switches from a broad sensitivity across a wide spectral range to a much narrower sensitivity centered around 490–500 nm on transition from marine to riverine conditions. These quantitative changes in visual pigment expression throughout the lifecycle may directly result from changes in the lighting conditions of the surrounding milieu.—Davies, W. L., Cowing, J. A., Carvalho, L. S., Potter, I. C., Trezise, A. E. O., Hunt, D. M., Collin, S. P. Functional characterization, tuning and regulation of visual pigment gene expression in an anadromous lamprey. FASEB J. 21, 2713–2724 (2007)

The origins of colour vision in vertebrates

The capacity for colour vision is mediated by the comparison of the signal intensities from photoreceptors of two or more types that differ in spectral sensitivity. Morphological, physiological and molecular analyses of the retina in an agnathan (jawless) fish, the lamprey Geotriu uwtrulis, may hold important clues to the origins of colour vision in vertebrates. Lampreys are extant representatives of an ancient group of vertebrates, the origins of which are thought to date back to at least the early Cambrian, approximately 540 million years ago. G. australis possesses five photoreceptor types, each with conelike ultrastructural features and different spectral sensitivities. Recent molecular genetic studies have also revealed that five visual pigment (opsin) genes are expressed in the retina, each of which is orthologous to the major classes of vertebrate opsin genes. These findings reveal that multiple opsin genes originated very early in vertebrate evolution, prior to the separation of the jawed and jawless vertebrate lineages, thereby providing the genetic basis for colour vision in all vertebrates.

Opsins: Evolution in Waiting

Complete vertebrate genome sequencing has revealed a remarkable stability and uniformity in the protein-coding gene set, which at first glance might suggest that gene duplication events are relatively rare. This may be a red herring, or at least a red cichlid, as the Lake Malawi cichlid fishes show rapid and extensive duplication and diversification of their retinal cone photoreceptor opsin genes.

Morphology, characterization, and distribution of retinal photoreceptors in the Australian lungfish Neoceratodus forsteri (Krefft, 1870)

The Australian lungfish Neoceratodus forsteri (Dipnoi) is an ancient fish that has a unique phylogenetic relationship among the basal Sarcopterygii. Here we examine the ultrastructure, histochemistry, and distribution of the retinal photoreceptors using a combination of light and electron microscopy in order to determine the characteristics of the photoreceptor layer in this living fossil. Similar proportions of rods (53%) and cones (47%) reveal that N. forsteri optimizes both scotopic and photopic sensitivity according to its visual demands. Scotopic sensitivity is optimized by a tapetum lucidum and extremely large rods (18.62 ± 2.68 μm ellipsoid diameter). Photopic sensitivity is optimized with a theoretical spatial resolving power of 3.28 ± 0.66 cycles degree−1, which is based on the spacing of at least three different cone types: a red cone containing a red oil droplet, a yellow cone containing a yellow ellipsoidal pigment, and a colorless cone containing multiple clear oil droplets. Topographic analysis reveals a heterogeneous distribution of all photoreceptor types, with peak cone densities predominantly found in temporal retina (6,020 rods mm−2, 4,670 red cones mm−2, 900 yellow cones mm−2, and 320 colorless cones mm−2), but ontogenetic changes in distribution are revealed. Spatial resolving power and the diameter of all photoreceptor types (except yellow cones) increases linearly with growth. The presence of at least three morphological types of cones provides the potential for color vision, which could play a role in the clearer waters of its freshwater environment. J. Comp. Neurol. 494:381–397, 2006.

Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes

The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interaction of c-Myb with the coactivator CBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore the molecular mechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction in myeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia.

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.