A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.
Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.
CL/P are very common birth defects in humans. The genetic mechanism underlying CL/P pathogenesis is poorly understood. MiRs, small non-coding RNAs that function to post-transcriptionally regulate gene expression, have been identified as pivotal modulators of various developmental events and diseases. To date, there is no individual miR or miR cluster that has been identified as functionally essential in mammalian CL/P. Here, we have discovered that deletion of miR-17-92 cluster in mice results in craniofacial malformations including CL/P. Importantly, MIR-17-92 is located on a critical human chromosome region associated with 13q deletion syndrome, a chromosomal disorder that presents with defects including CL/P, suggesting the advantages of our animal model to study human disease. Moreover, our work demonstrated that miR-17-92 cluster directly repressed T-box factors, which have critical functions during craniofacial development. We further showed that miR-17-92 was directly activated by Bmp-signaling and transcription factor AP-2α. Together, our work identified a novel miR-mediated transcriptional network underlying CL/P, providing new insights into craniofacial developmental biology.
With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H♂)×healthy females (H♀); ii) infected males (I♂)×healthy females (H♀); iii) healthy males (H♂)×infected females (I♀) and iv) infected males (I♂)×infected females (I♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.
A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large-deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origins of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. The homology of nucleotides in the deleted regions was as high as 79%, similar to those of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that independently generates and sustains deletion mutants within each virus population. Functional analyses using different proportions of complete and deletion genotypes were performed with the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete/deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but there were no significant changes in the speed of kill. In contrast, OB yields increased only in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most extensively deleted SeMNPV genotype and contribute toward the elucidation of the role of such mutants in baculovirus populations.
We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2β and AP-2γ, showed expression patterns similar to AP-2α.
Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2β are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/β-immunopositive population. To address possible redundant roles for AP-2α and AP-2β during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone.
These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.
AP-2 transcription factors; retinal development; horizontal cell; amacrine cell; Tcfap2a/b double mutant; redundant roles
Survival rates for patients with idiopathic pulmonary arterial hypertension (IPAH) have improved with the introduction of PAH-specific therapies. However, the time between patient-reported onset of symptoms and a definitive diagnosis of IPAH is consistently delayed. We conducted a retrospective, multi-center, descriptive investigation in order to (a) understand what factors contribute to persistent diagnostic delays, and (b) examine the time from initial symptom onset to a definitive diagnosis of IPAH. Between January 2007 and December 2008, we enrolled consecutively diagnosed adults with IPAH from four tertiary referral centers in Australia. Screening of patient records and “one-on-one” interviews were used to determine the time from patient-described initial symptoms to a diagnosis of IPAH, confirmed by right heart catheterization (RHC). Thirty-two participants (69% female) were studied. Mean age at symptom onset was 56 ± 16.4 years and 96% reported exertional dyspnea. Mean time from symptom onset to diagnosis was 47 ± 34 months with patients subsequently aged 60 ± 17.3 years. Patients reported 5.3 ± 3.8 GP visits and 3.0 ± 2.1 specialist reviews before being seen at a pulmonary hypertension (PH) center. Advanced age, number of general practitioner (GP) visits, heart rate, and systolic blood pressure at the time of diagnosis were significantly associated with the observed delay. We found a significant delay of 3.9 years from symptom onset to a diagnosis of IPAH in Australia. Exertional dyspnea is the most common presenting symptom. Current practice within Australia does not appear to have the specific capacity for timely, multi-factorial evaluation of breathlessness and potential IPAH.
diagnosis; epidemiology; pulmonary hypertension; pulmonary arterial hypertension
Invertebrate and vertebrate vestigial (vg) and vestigial-like (vgl) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the TEAD/Scalloped family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton.
Vestigial-like; Vgl-2; VITO-1; craniofacial; zebrafish; FGF; Retinoic Acid; Cell Death
signaling pathways and transcriptional effectors responsible for directing
mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens.
We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm.
Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death.
Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.
This study shows the ability of the lens to respond to altered Smoothened activity. Constitutive activation of Smoothened in the surface ectoderm and derivatives, including the lens results in altered expression of lens markers and abnormal lens differentiation and morphology.
Morphogenesis of mammalian facial processes requires coordination of cellular proliferation, migration, and apoptosis to develop intricate features. Cleft lip and/or palate (CL/P), the most frequent human craniofacial birth defect, can be caused by perturbation of any of these programs. Mutations of WNT, P63, and IRF6 yield CL/P in humans and mice; however, how these genes are regulated remains elusive. We generated mouse lines lacking Pbx genes in cephalic ectoderm and demonstrated that they exhibit fully penetrant CL/P and perturbed Wnt signalling. We also characterized a midfacial regulatory element that Pbx proteins bind in order to control the expression of Wnt9b-Wnt3, which in turn regulate p63. Altogether, we establish a Pbx-dependent Wnt-p63-Irf6 regulatory module in midfacial ectoderm that is conserved within mammals. Dysregulation of this network leads to localized suppression of midfacial apoptosis and CL/P. Ectopic Wnt ectodermal expression in Pbx mutants rescues the clefting, opening avenues for tissue repair.
Face; Ectoderm; Lambdoidal Junction; Pbx; Wnt; p63; Irf6; Wnt midfacial enhancer; CL/P; Mouse
Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.
Craniofacial anomalies account for approximately one third of all birth defects, and holoprosencephaly (HPE) is the most common structural malformation of the embryonic brain. HPE is a failure of the forebrain to bifurcate and is a heterogeneous disorder that is often found in patients together with other craniofacial malformations. Currently, it is not known if these phenotypes arise through a common etiology and pathogenesis, as the genetic lesions responsible for HPE have only been identified in about 20% of affected individuals. Here we demonstrate for the first time that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which highlights the importance of Hh signaling in complex craniofacial disorders.
Car tires are important habitats for mosquito development because of the high density populations they can harbor and their presence in urban settings. Water in experimental tires was treated with one of three insecticides or an untreated control. Aquatic invertebrates were sampled at weekly intervals. Eggs, larval and pupal samples were laboratory-reared to estimate seasonal fluctuations in Aedes aegypti and Ae. albopictus abundance.
Spinosad treatments at 1 or 5 ppm (mg a.i./liter) provided 6–8 weeks of effective control of Ae. aegypti, Ae. albopictus, Culex quinquefasiatus and Cx. coronator larvae, both in the dry season and the rainy season when mosquito populations increased markedly in southern Mexico. Spinosad continued to provide partial control of larvae for several weeks after initial recolonization of treated tires. The larvicidal performance of VectoBac 12AS (Bacillus thuringiensis var. israelensis) was relatively poor with one week of complete control of Aedes spp. larvae and no discernible control of Culex spp., whereas the duration of larvicidal activity of 1% temephos mineral-based granules was intermediate between those of VectoBac and spinosad treatments. Populations of chironomids, ostracods and Toxorhynchites theobaldi were generally reduced in spinosad and temephos treatments, but were similar in control and VectoBac treatments.
The present study is the first to report spinosad as an effective larvicide against Cx. coronator, which is currently invading the southern United States. These results substantiate the use of spinosad as a highly effective mosquito larvicide, even in habitats such as unused car tires that can represent prolific sources of adult mosquitoes.
In mice, BDNF provided by the developing taste epithelium is required for gustatory neuron survival following target innervation. However, we find that expression of BDNF, as detected by BDNF-driven β-galactosidase, begins in the cranial ganglia before its expression in the central (hindbrain) or peripheral (taste papillae) targets of these sensory neurons, and before gustatory ganglion cells innervate either target. To test early BDNF function, we examined the ganglia of bdnf null mice before target innervation, and found that while initial neuron survival is unaltered, early neuron development is disrupted. In addition, fate mapping analysis in mice demonstrates that murine cranial ganglia arise from 2 embryonic populations, i.e., epibranchial placodes and neural crest, as has been described for these ganglia in non-mammalian vertebrates. Only placodal neurons produce BDNF, however, which indicates that prior to innervation, early ganglionic BDNF produced by placode-derived cells promotes gustatory neuron development.
BDNF; cranial ganglia; maturation; gustatory; placode; neural crest; neuron; mouse; cre recombinase; fate mapping; ROSA reporter line
The canonical Wnt/β-catenin pathway is an essential component of multiple developmental processes. To investigate the role of this pathway in the ectoderm during facial morphogenesis, we generated conditional β-catenin mouse mutants using a novel ectoderm-specific Cre recombinase transgenic line. Our results demonstrate that ablating or stabilizing β-catenin in the embryonic ectoderm causes dramatic changes in facial morphology. There are accompanying alterations in the expression of Fgf8 and Shh, key molecules that establish a signaling center critical for facial patterning, the frontonasal ectodermal zone (FEZ). These data indicate that Wnt/β-catenin signaling within the ectoderm is critical for facial development and further suggest that this pathway is an important mechanism for generating the diverse facial shapes of vertebrates during evolution.
craniofacial; ectoderm; Wnt/β-catenin; FEZ; FGF8
Sublethal infections by Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) are common in field populations of the beet armyworm (S. exigua, Hübner) in the Almerian horticultural region of Spain. Inoculation of second, third, and fourth instars with occlusion bodies (OBs) of an isolate (VT-SeAl1) associated with vertically transmitted infections resulted in 15 to 100% of sublethal infection in adult survivors, as determined by reverse transcription-PCR (RT-PCR) detection of viral DNA polymerase transcripts, and quantitative PCR (qPCR) targeted at the DNA polymerase gene. The prevalence of adult sublethal infection was positively related to the inoculum OB concentration consumed during the larval stage. Sublethal infections persisted in OB-treated insects for at least five generations. Viral transcripts were more frequently detected in adult insects than in third instars. qPCR analysis indicated a consistently higher prevalence of sublethal infection than RT-PCR. Sublethal infection was associated with significant reductions in pupal weight, adult emergence, fecundity, and fertility (egg hatch) and significant increases in larval development time and duration of the preoviposition period. Insects taken from a persistently infected experimental population were significantly more susceptible to the OB inoculum than control insects that originated from the same virus-free colony as the persistently infected insects. We conclude that OB treatment results in rapid establishment of sublethal infections that persist between generations and which incur costs in the development and reproductive capacity of the host insect.
Appropriate development of the retina and optic nerve requires that the forebrain-derived optic neuroepithelium undergoes a precisely coordinated sequence of patterning and morphogenetic events, processes which are highly influenced by signals from adjacent tissues. Our previous work has suggested that transcription factor activating protein-2 alpha (AP-2α; Tcfap2a) has a non-cell autonomous role in optic cup (OC) development; however, it remained unclear how OC abnormalities in AP-2α knockout (KO) mice arise at the morphological and molecular level. In this study, we show that patterning and morphogenetic defects in the AP-2α KO optic neuroepithelium begin at the optic vesicle stage. During subsequent OC formation, ectopic neural retina and optic stalk-like tissue replaced regions of retinal pigment epithelium. AP-2α KO eyes also displayed coloboma in the ventral retina, and a rare phenotype in which the optic stalk completely failed to extend, causing the OCs to be drawn inward to the midline. We detected evidence of increased sonic hedgehog signaling in the AP-2α KO forebrain neuroepithelium, which likely contributed to multiple aspects of the ocular phenotype, including expansion of PAX2-positive optic stalk-like tissue into the OC. Our data suggest that loss of AP-2α in multiple tissues in the craniofacial region leads to severe OC and optic stalk abnormalities by disturbing the tissue–tissue interactions required for ocular development. In view of recent data showing that mutations in human TFAP2A result in similar eye defects, the current findings demonstrate that AP-2α KO mice provide a valuable model for human ocular disease.
Previous studies identified Inka1 as a gene regulated by AP-2α in the neural crest required for craniofacial morphogenesis in fish and frog. Here, we extend the analysis of Inka1 function and regulation to the mouse by generating a LacZ knock-in allele. Inka1-LacZ allele expression occurs in the cephalic mesenchyme, heart, and paraxial mesoderm prior to E8.5. Subsequently, expression is observed in the migratory neural crest cells and their derivatives. Consistent with expression of Inka1 in tissues of the developing head during neurulation, a low percentage of Inka1−/− mice show exencephaly while the remainder are viable and fertile. Further studies indicate that AP-2α is not required for Inka1 expression in the mouse, and suggest that there is no significant genetic interaction between these two factors during embryogenesis. Together, these data demonstrate that while the expression domain of Inka1 is conserved among vertebrates, its function and regulation are not.
neural crest; AP-2α; Inka1; neural tube closure; PAK4
An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission.
baculovirus; co-occlusion; genotype diversity; occlusion body; transmission; virion
Within the olfactory bulb, neurons and their axonal connections are organized into distinct layers corresponding to different functionalities. Here we demonstrate that transcription factor AP-2ε is required for olfactory bulb development, specifically the establishment of appropriate neuronal lamination. During normal mouse embryogenesis, AP-2ε transcripts are one of the earliest markers of olfactory bulb formation, and expression eventually becomes refined to the projection neurons, the mitral and tufted cells. To assess the function of AP-2ε in olfaction, we generated a null allele (the “AK” allele) by inserting a Cre recombinase transgene into the endogenous AP-2ε genomic locus. AP-2ε-null mice exhibited defective olfactory bulb architecture. The mitral cell layer was disorganized, typified by misoriented and aberrantly positioned projection neurons, and the adjacent internal plexiform layer was absent. Despite the significant disruption of olfactory bulb organization, AP-2ε null mice were viable and could discriminate a variety of odors. AP-2ε-null mice thus provide compelling evidence for the robust nature of the mouse olfactory system, and serve as a model system to probe both the regulation of neuronal lamination and the functional circuitry of the olfactory bulb. We also show that Cre recombinase expression directed by the AP-2ε locus can specifically target floxed genes within the olfactory bulb, extending the utility of this AK allele.
AP-2ε; Tcfap2e; olfactory bulb; Cre recombinase; mitral cell; internal plexiform layer
In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses.
We used Mass spectrometry-based proteomics (MSP) to identify and quantify thousands of proteins from healthy and collapsing bee colonies. MSP revealed two unreported RNA viruses in North American honey bees, Varroa destructor-1 virus and Kakugo virus, and identified an invertebrate iridescent virus (IIV) (Iridoviridae) associated with CCD colonies. Prevalence of IIV significantly discriminated among strong, failing, and collapsed colonies. In addition, bees in failing colonies contained not only IIV, but also Nosema. Co-occurrence of these microbes consistently marked CCD in (1) bees from commercial apiaries sampled across the U.S. in 2006–2007, (2) bees sequentially sampled as the disorder progressed in an observation hive colony in 2008, and (3) bees from a recurrence of CCD in Florida in 2009. The pathogen pairing was not observed in samples from colonies with no history of CCD, namely bees from Australia and a large, non-migratory beekeeping business in Montana. Laboratory cage trials with a strain of IIV type 6 and Nosema ceranae confirmed that co-infection with these two pathogens was more lethal to bees than either pathogen alone.
These findings implicate co-infection by IIV and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. We next need to characterize the IIV and Nosema that we detected and develop management practices to reduce honey bee losses.
The stabilities of the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) complete genome bacmid (Sfbac) and a deletion recombinant (Sf29null) in which the Sf29 gene was replaced by a kanamycin resistance cassette were determined during sequential rounds of per os infection in insect larvae. The Sf29 gene is a viral factor that determines the number of virions in occlusion bodies (OBs). The Sf29null bacmid virus was able to recover the Sf29 gene during passage. After the third passage (P3) of Sf29null bacmid OBs, the population was observed to reach an equilibrium involving a mixture of those with a kanamycin resistance cassette and those with the Sf29 gene. The biological activity of Sf29null bacmid OBs at P3 was similar to that of Sfbac OBs. The recovered gene in the Sf29null virus was 98 to 100% homologous to the Sf29 genes of different SfMNPV genotypes. Reverse transcription-PCR analysis of uninoculated S. frugiperda larvae confirmed the expression of the SfMNPV ie-0 and Sf29 genes, indicating that the insect colony harbors a covert SfMNPV infection. Additionally, the nonessential bacterial artificial chromosome vector was spontaneously deleted from both viral genomes upon passage in insects.
Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions – the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5–E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional transcription units that likely share cis-acting sequences with well-characterized genes. Overall, our studies provide a valuable resource for probing orofacial development and a robust dataset for bioinformatic analysis of spatial and temporal gene expression changes during embryogenesis.
The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-BΔ16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-BΔpifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-BΔ16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-BΔ16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-BΔpifs were almost identical to the results observed with SfNIC-BΔ16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-BΔ16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of ∼85% SfNIC-B plus ∼15% SfNIC-BΔ16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNIC-BΔpifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1- and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.
The AP-2 transcription factor family is linked with development of the head and limbs in both vertebrate and invertebrate species. Recent evidence has also implicated this gene family in the evolution of the neural crest in chordates, a critical step that allowed the development and elaboration of the vertebrate craniofacial skeleton. In mice, the inappropriate embryonic expression of one particular AP-2 gene, Tcfap2a - encoding AP-2α - results in multiple developmental abnormalities, including craniofacial and limb defects. Thus, Tcfap2a provides a valuable genetic resource to analyze the regulatory hierarchy responsible for the evolution and development of the face and limbs. Previous studies have identified a 2 kilobase intronic region of both the mouse and human AP-2α locus that directs expression of a linked LacZ transgene to the facial processes and the distal mesenchyme of the limb bud in transgenic mice. Further analysis identified two highly conserved regions of ~ 200-400 bp within this tissue-specific enhancer. We have now initiated a transgenic and biochemical analysis of the most important of these highly conserved regions. Our analysis indicates that although the sequences regulating face and limb expression have been integrated into a single enhancer, different cis-acting sequences ultimately control these two expression domains. Moreover, these studies demonstrate that a conserved STAT binding site provides a major contribution to the expression of Tcfap2a in the facial prominences.
Tcfap2a; limb bud; frontal nasal process; STAT1; Sox6
The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2γ is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2γ which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2γ in skin development. Mice deficient for AP-2γ exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2γ in skin development, and reveal the existence of regulatory factors that can compensate for AP-2γ in its absence.
AP-2; Tcfap2c; skin; epidermis; keratinocyte; filaggrin; keratin; differentiation; embryo development
The mechanisms by which mammalian epidermal stem cells cease to proliferate and embark upon terminal differentiation are still poorly understood. By conditionally ablating two highly expressed transcription factors, AP-2α and AP-2γ, we unmasked functional redundancies and discovered an essential role for AP-2s in the process. In vivo and in vitro, AP-2 deficiency is accompanied by surprisingly minimal changes in basal gene expression but severely perturbed terminal differentiation and suppression of additional transcription factors and structural genes involved. In dissecting the underlying molecular pathways, we uncover parallel pathways involving AP-2 and Notch signaling, which converge to govern CCAAT/enhancer binding protein genes and orchestrate the transition from basal proliferation to suprabasal differentiation. Finally, we extend the striking similarities in compromising either Notch signaling or AP-2α/AP-2γ in developing skin to that in postnatal skin, where all hair follicles and sebaceous gland differentiation are also repressed and overt signs of premalignant conversion emerge.