Same but different: ontogeny and evolution of the Musculus adductor mandibulae in the Tetraodontiformes.

The morphological diversity of fishes provides a rich source to address questions regarding the evolution of complex and novel forms. The Tetraodontiformes represent an order of highly derived teleosts including fishes, such as the pelagic ocean sunfishes, triggerfishes, and pufferfishes. This makes the order attractive for comparative analyses to understand the role of development in generating new forms during evolution. The adductor mandibulae complex, the main muscle associated with jaw closure, represents an ideal model system within the Tetraodontiformes. The adductor mandibulae differs in terms of partitions and their attachment sites between members of the different tetraodontiform families. In order to understand the evolution of the jaws among the Tetraodontiformes, we investigate the development of the adductor mandibulae in pufferfishes and triggerfishes as representatives of two different suborders (Balistoidei and Tetraodontoidei) that follows two different adaptations to a durophagous feeding mode. We show that the varied patterns of the adductor mandibulae derive from similar developmental sequence of subdivision of the partitions. We propose a conserved developmental program for partitioning of the adductor mandibulae as a foundation for the evolution of different patterns of subdivisions in Tetraodontiformes. Furthermore, we argue that derived conditions in the higher taxa are realized by supplementary subdivisions and altered attachment sites. These findings support a reinterpretation of homology of different muscle partitions among the Tetraodontiformes, as muscle partitions previously thought to be disparate, are now clearly related.

Hemispheric asymmetry in ocean change and the productivity of ecosystem sentinels.

Climate change and other human activities are causing profound effects on marine ecosystem productivity. We show that the breeding success of seabirds is tracking hemispheric differences in ocean warming and human impacts, with the strongest effects on fish-eating, surface-foraging species in the north. Hemispheric asymmetry suggests the need for ocean management at hemispheric scales. For the north, tactical, climate-based recovery plans for forage fish resources are needed to recover seabird breeding productivity. In the south, lower-magnitude change in seabird productivity presents opportunities for strategic management approaches such as large marine protected areas to sustain food webs and maintain predator productivity. Global monitoring of seabird productivity enables the detection of ecosystem change in remote regions and contributes to our understanding of marine climate impacts on ecosystems.

Influence of wing loading on the trade-off between pursuit-diving and flight in common guillemots and razorbills.

Species of bird that use their wings for underwater propulsion are thought to face evolutionary trade-offs between flight and diving, leading to the prediction that species with different wing areas relative to body mass (i.e. different wing loadings) also differ in the relative importance of flight and diving activity during foraging trips. We tested this hypothesis for two similarly sized species of Alcidae (common guillemots and razorbills) by using bird-borne devices to examine three-dimensional foraging behaviour at a single colony. Guillemots have 30% higher wing loading than razorbills and, in keeping with this difference, razorbills spent twice as long in flight as a proportion of trip duration whereas guillemots spent twice as long in diving activity. Razorbills made a large number of short, relatively shallow dives and spent little time in the bottom phase of the dive whereas guillemots made fewer dives but frequently attained depths suggesting that they were near the seabed (ca. 35-70 m). The bottom phase of dives by guillemots was relatively long, indicating that they spent considerable time searching for and pursuing prey. Guillemots also spent a greater proportion of each dive bout underwater and had faster rates of descent, indicating that they were more adept at maximising time for pursuit and capture of prey. These differences in foraging behaviour may partly reflect guillemots feeding their chicks single large prey obtained near the bottom and razorbills feeding their chicks multiple prey from the water column. Nonetheless, our data support the notion that interspecific differences in wing loadings of auks reflect an evolutionary trade-off between aerial and underwater locomotion.

Modeling survival at multi-population scales using mark-recapture data.

The demography of vertebrate populations is governed in part by processes operating at large spatial scales that have synchronizing effects on demographic parameters over large geographic areas, and in part, by local processes that generate fluctuations that are independent across populations. We describe a statistical model for the analysis of individual monitoring data at the multi-population scale that allows us to (1) split up temporal variation in survival into two components that account for these two types of processes and (2) evaluate the role of environmental factors in generating these two components. We derive from this model an index of synchrony among populations in the pattern of temporal variation in survival, and we evaluate the extent to which environmental factors contribute to synchronize or desynchronize survival variation among populations. When applied to individual monitoring data from four colonies of the Atlantic Puffin (Fratercula arctica), 67% of between-year variance in adult survival was accounted for by a global spatial-scale component, indicating substantial synchrony among colonies. Local sea surface temperature (SST) accounted for 40% of the global spatial-scale component but also for an equally large fraction of the local-scale component. SST thus acted at the same time as both a synchronizing and a desynchronizing agent. Between-year variation in adult survival not explained by the effect of local SST was as synchronized as total between-year variation, suggesting that other unknown environmental factors acted as synchronizing agents. Our approach, which focuses on demographic mechanisms at the multi-population scale, ideally should be combined with investigations of population size time series in order to characterize thoroughly the processes that underlie patterns of multi-population dynamics and, ultimately, range dynamics.

A role for G protein-coupled receptor 137b in bone remodeling in mouse and zebrafish.

G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba-deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba-/- mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba-deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba-/- fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation.

The demographic impact of extreme events: stochastic weather drives survival and population dynamics in a long-lived seabird.

1. Most scenarios for future climate change predict increased variability and thus increased frequency of extreme weather events. To predict impacts of climate change on wild populations, we need to understand whether this translates into increased variability in demographic parameters, which would lead to reduced population growth rates even without a change in mean parameter values. This requires robust estimates of temporal process variance, for example in survival, and identification of weather covariates linked to interannual variability. 2. The European shag Phalacrocorax aristotelis (L.) shows unusually large variability in population size, and large-scale mortality events have been linked to winter gales. We estimated first-year, second-year and adult survival based on 43 years of ringing and dead recovery data from the Isle of May, Scotland, using recent methods to quantify temporal process variance and identify aspects of winter weather linked to survival. 3. Survival was highly variable for all age groups, and for second-year and adult birds process variance declined strongly when the most extreme year was excluded. Survival in these age groups was low in winters with strong onshore winds and high rainfall. Variation in first-year survival was not related to winter weather, and process variance, although high, was less affected by extreme years. A stochastic population model showed that increasing process variance in survival would lead to reduced population growth rate and increasing probability of extinction. 4. As in other cormorants, shag plumage is only partially waterproof, presumably an adaptation to highly efficient underwater foraging. We speculate that this adaptation may make individuals vulnerable to rough winter weather, leading to boom-and-bust dynamics, where rapid population growth under favourable conditions allows recovery from periodic large-scale weather-related mortality. 5. Given that extreme weather events are predicted to become more frequent, species such as shags that are vulnerable to such events are likely to exhibit stronger reductions in population growth than would be expected from changes in mean climate. Vulnerability to extreme events thus needs to be accounted for when predicting the ecological impacts of climate change.

Renin and renin mRNA in proximal tubules of the rat kidney.

The present study was undertaken to assess the presence of renin enzymatic activity and renin mRNA in proximal tubules of rat kidneys, and to determine the effect of converting enzyme inhibition (CEI) on proximal tubule renin gene expression. Proximal convoluted tubules (PCT), proximal straight tubules (PST), outer medullary collecting ducts (OMCD), and glomeruli (Gloms) were isolated by microdissection. Renin activity was measured in sonicated segments by radioimmunoassay. Renin mRNA levels were assessed using a quantitative PCR. Renin activity in PCT averaged 51 +/- 15 microGU/mm compared to 405 +/- 120 microGU/glomerulus. No measurable renin activity was found in PST and OMCD. Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and 7.5. Renin mRNA was consistently detectable in cDNA prepared from PCT and PST, although its abundance per mm tubule was about 1/500th that found in one glomerulus. Renin mRNA was not detectable in OMCD. Tubular renin PCR product identity was confirmed by restriction digestion. CEI administration increased glomerular renin activity and renin mRNA, but not proximal tubular renin. The absence of a stimulatory effect of CEI on proximal tubule renin gene expression suggests the operation of different intracellular signals in control of renin synthesis in the proximal tubule than in the vascular compartment.

Small teleost fish provide new insights into human skeletal diseases.

Small teleost fish such as zebrafish and medaka are increasingly studied as models for human skeletal diseases. Efficient new genome editing tools combined with advances in the analysis of skeletal phenotypes provide new insights into fundamental processes of skeletal development. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. Several unique features of the skeleton relate to the extremely small size of early fish embryos and the small size of adult fish used as models. A detailed analysis of the plethora of interesting skeletal phenotypes in zebrafish and medaka pushes available skeletal imaging techniques to their respective limits and promotes the development of new imaging techniques. Impressive numbers of zebrafish and medaka mutants with interesting skeletal phenotypes have been characterized, complemented by transgenic zebrafish and medaka lines. The advent of efficient genome editing tools, such as TALEN and CRISPR/Cas9, allows to introduce targeted deficiencies in genes of model teleosts to generate skeletal phenotypes that resemble human skeletal diseases. This review will also discuss other attractive aspects of the teleost skeleton. This includes the capacity for lifelong tooth replacement and for the regeneration of dermal skeletal elements, such as scales and fin rays, which further increases the value of zebrafish and medaka models for skeletal research.

p53-mediated accumulation of hypophosphorylated pRb after the G1 restriction point fails to halt cell cycle progression.

This study analyses whether the inability of p53 to induce G1 arrest after the restriction point relates to an inability to modulate pRb phosphorylation. Transient p53 overexpression in normal human diploid fibroblasts and p53-deficient cancer cells led to increased levels of the cyclin-dependent kinase inhibitor p21 cip1/Waf1/Sdi1 and an accumulation of hypophosphorylated pRb in cells growing asynchronously and in cells synchronized in late G1 or M. Similarly, gamma-irradiation of asynchronous, late-G1, or S phase fibroblasts led to an increase in hypophosphorylated pRb. Experiments with fibroblasts expressing the HPV16 E6 protein indicated that accumulation of hypophosphorylated pRb required functional p53. Progression into and through S phase was not altered by the presence of hypophosphorylated pRb in late G1, consistent with the failure of p53 to mediate G1 arrest in cells that are past the restriction point. These data indicate that accumulation of hypophosphorylated pRb has significantly different effects on cell cycle progression in early G1 versus late G1 or S phase.

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro.

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.

Myristoylation-dependent binding of HIV-1 Nef to CD4.

The nef gene is conserved throughout the primate lentivirus family. Although dispensable in vitro, an important role for nef in vivo is suggested by the failure of SIV nef mutants to establish persistent viraemia. Although the biochemical function of the Nef protein remains equivocal, a consistent theme has emerged with the reproducible observation that Nef expression results in the down-modulation of the cell surface marker CD4. Down-modulation requires amino acid sequences within the cytoplasmic domain of CD4 but occurs by a mechanism distinct from the normal serine phosphorylation-dependent pathway. As CD4 is a transmembrane glycoprotein and Nef a myristoylated protein targeted to the cytoplasmic face of the plasma membrane we considered that a direct interaction between Nef and CD4 might play a role in down-modulation. Here we demonstrate that a baculovirus-expressed Nef-GST fusion protein interacts specifically with CD4. This interaction requires co-expression in the same cell and is dependent on Nef myristoylation. The site of Nef interaction maps to the cytoplasmic domain of CD4, as a deletion mutant lacking this domain fails to interact with Nef. This observation sheds new light on the biochemical function of Nef and offers new opportunities for the future development of HIV chemotherapy.

Induction of activator protein 1 (AP-1) in macrophages by human immunodeficiency virus type-1 NEF is a cell-type-specific response that requires both hck and MAPK signaling events.

Human immunodeficiency virus type 1 (HIV-1) Nef is important for viral infectivity and pathogenicity. HIV-1 infection is associated with inappropriate activation and defects in the function of monocytes/macrophages. We have studied the effects of HIV-1 Nef in the murine (RAW264.7) and human (THP-1) monocyte-macrophage cell lines. Investigation of the activator protein-1 (AP-1) transcription factor showed that Nef expression induced both its DNA binding and transcriptional activities. Increased AP-1 DNA binding activity in RAW264.7 cells was associated with raised levels of c-Fos expression and induction of mRNA for the AP-1 responsive tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. Mutagenesis and kinase inhibition studies were employed to determine signaling pathways used by Nef to induce AP-1. Data from these studies indicated that induction of AP-1 by Nef is likely to be mediated through the MAPK (ERK1 and 2) signaling pathway and requires the proline-rich PxxP motif of Nef, suggesting the involvement of upstream protein kinases belonging to the Src family. Effects of Nef on AP-1 induction were cell lineage-specific, being stimulatory in macrophages, inhibitory in T cells and without effect in HeLa cells. These latter two observations led us to test the possibility that cell-specific interactions of Nef with Src family proteins may modulate AP-1 activity. To this end we demonstrated that a dominant-negative Hck mutant caused inhibition of Nef-mediated AP-1 DNA binding activity in RAW cells. In conclusion, induction of AP-1 by Nef is a specific feature of human and murine macrophage cell lines that requires signal transduction events involving Hck and MAPKs.

p53 gene therapy in a rat model of hepatocellular carcinoma: intra-arterial delivery of a recombinant adenovirus.

p53 tumor suppressor gene therapy has been proposed for cancers characterized by inactivation of p53 function, and successful therapy will require efficient strategies for gene delivery. To maximize transgene expression in tumors, a clinical strategy has been proposed to treat neoplasms in the liver via hepatic artery administration of a recombinant adenovirus encoding wild-type p53 (rAd-p53). We have developed a syngeneic rat model using a p53mut hepatocellular carcinoma cell line (McA-RH7777) that results in multifocal liver tumor nodules to provide experimental support for this strategy. Treatment of McA-RH7777 cells with rAd-p53 in vitro resulted in efficient transgene expression, growth suppression, and apoptosis. Intrahepatic artery dosing with rAd-p53 or an adenovirus encoding beta-galactosidase (rAd-betagal) increased transgene expression in tumor tissue and decreased systemic exposure when compared with i.v. dosing. Daily hepatic artery dosing of rAd-p53 suppressed tumor growth when compared with untreated rats or animals treated with rAd-betagal. These data demonstrate the potential for arterial gene delivery to tumors using recombinant adenoviruses, and support continued investigation of rAd-p53 gene therapy for liver malignancies.

Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer.

We have constructed recombinant human adenoviruses that express wild-type human p53 under the control of either the Ad 2 major late promoter (MLP) or the human cytomegalovirus (CMV) immediate early gene promoter. Each construct replaces the Ad 5 E1a and E1b coding sequences necessary for viral replication with the p53 cDNA and MLP or CMV promoter. These p53/Ad recombinants are able to express p53 protein in a dose-dependent manner in infected human cancer cells. Tumor suppressor activity of the expressed p53 protein was assayed by several methods. [3H]Thymidine incorporation assays showed that the recombinant adenoviruses were capable of inhibiting DNA synthesis in a p53-specific, dose-dependent fashion. Ex vivo treatment of Saos-2 tumor cells, followed by injection of the treated cells into nude mice, led to complete tumor suppression using the MLP/p53 recombinant. Following a single injection of CMV/p53 recombinant adenovirus into the peritumoral space surrounding an in vivo established tumor derived from a human small cell lung carcinoma cell line (NIH-H69), we were able to detect p53 mRNA in the tumors at 2 and 7 days post-injection. Continued treatment of established H69 tumors with MLP/p53 recombinant led to reduced tumor growth and increased survival time compared to control treated animals. These results indicate that recombinant adenoviruses expressing wild-type p53 may be useful vectors for gene therapy of human cancer.

Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein.

Human malignancies are often characterized by mutations of the p53 tumor suppressor gene. In a large proportion of cases, the mutation results in production of an altered protein that can bind and inactivate the wild-type gene product. This "dominant-negative" activity of mutant p53 molecules may limit the utility of p53 gene therapy of cancer. Using replication-deficient recombinant adenoviruses (rAd-p53) as a p53 gene delivery system, we evaluated the effects of p53 reintroduction on a series of 45 human cell lines containing wild-type, mutated, or no p53 protein. Results indicate a p53-specific, dose-dependent, and promoter-specific growth inhibition of a majority of p53-altered cell lines that correlates with the degree of adenovirus transgene expression. Similar effects were not observed on cells containing wild-type p53. rAd-p53 inhibited the growth of cells expressing various mutant p53 proteins including those characterized as "dominant negative mutants", and the antiproliferative effects were not abrogated by high levels of endogenous mutated p53 protein. In vivo, rAd-p53 also suppressed tumor growth and increased survival of nude mice bearing tumors that express mutant p53. These results support a role for p53 gene therapy of cancer, including malignancies harboring mutations in this tumor suppressor gene.

Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene.

We have investigated whether adenovirally mediated gene transfer of the herpes simplex thymidine kinase gene to human hepatocellular carcinoma (HCC) cell lines can sensitize these cells to the prodrug ganciclovir and thereby provide a therapeutic option for this intractable cancer. Two replication-deficient adenoviruses encoding for the herpes simplex virus type-1 (HSV) thymidine kinase (TK) gene were generated in which expression of TK is under the control of either the human cytomegalovirus immediate early promoter (CMV) or the human alpha-fetoprotein (AFP) promoter/enhancer. We demonstrate that the combination of adenovirally mediated TK gene transfer and ganciclovir treatment effectively inhibits proliferation and causes cell death of HCC cells in vitro and that in vivo TK gene transfer and ganciclovir treatment inhibits hepatocellular tumor growth in a mouse model of this cancer. Furthermore, we show that expression of the TK gene can be restricted to those HCCs that express the tumor marker AFP through the incorporation of the AFP enhancer/promoter within an adenoviral vector.

A recombinant adenoviral vector expressing full-length human retinoblastoma susceptibility gene inhibits human tumor cell growth.

As a prelude to considering retinoblastoma (RB) gene therapy for cancer, a series of human tumor cell lines with either full-length, mutated, or undetectable RB protein were treated with recombinant adenovirus encoding RB (ACNRB). Both RB protein expression and the cytotoxic and antiproliferative effects of ACNRB treatment were evaluated. While the transgene expression of a reporter virus encoding the beta-galactosidase enzyme (rAd-beta-gal) varied among cell lines, the reintroduction and expression of the RB gene resulted in a pronounced inhibition of cellular proliferation in RB-altered cell lines. An antiproliferative response was observed with control adenovirus treatment in some cell lines. ACNRB treatment did not cause detectable cytotoxicity in either RB+ or RB-altered cells. Dose-dependent cytostasis was observed in RB- cell lines. In vivo tumor suppression was observed in a breast xenograft model subsequent to the treatment of established tumors with ACNRB. These data support a role for RB gene therapy of tumors with RB mutations and provide a basis for the further evaluation of ACNRB gene therapy of human cancer.

Fish is Fish: the use of experimental model species to reveal causes of skeletal diversity in evolution and disease.

Fishes are wonderfully diverse. This variety is a result of the ability of ray-finned fishes to adapt to a wide range of environments, and has made them more specious than the rest of vertebrates combined. With such diversity it is easy to dismiss comparisons between distantly related fishes in efforts to understand the biology of a particular fish species. However, shared ancestry and the conservation of developmental mechanisms, morphological features and physiology provide the ability to use comparative analyses between different organisms to understand mechanisms of development and physiology. The use of species that are amenable to experimental investigation provides tools to approach questions that would not be feasible in other 'non-model' organisms. For example, the use of small teleost fishes such as zebrafish and medaka has been powerful for analysis of gene function and mechanisms of disease in humans, including skeletal diseases. However, use of these fish to aid in understanding variation and disease in other fishes has been largely unexplored. This is especially evident in aquaculture research. Here we highlight the utility of these small laboratory fishes to study genetic and developmental factors that underlie skeletal malformations that occur under farming conditions. We highlight several areas in which model species can serve as a resource for identifying the causes of variation in economically important fish species as well as to assess strategies to alleviate the expression of the variant phenotypes in farmed fish. We focus on genetic causes of skeletal deformities in the zebrafish and medaka that closely resemble phenotypes observed both in farmed as well as natural populations of fishes.