Efficiency Correction Is Required for Accurate Quantitative PCR Analysis and Reporting.

BACKGROUND: Quantitative PCR (qPCR) aims to measure the DNA or RNA concentration in diagnostic and biological samples based on the quantification cycle (Cq) value observed in the amplification curves. Results of qPCR experiments are regularly calculated as if all assays are 100% efficient or reported as just Cq, ΔCq, or ΔΔCq values. CONTENTS: When the reaction shows specific amplification, it should be deemed to be positive, regardless of the observed Cq. Because the Cq is highly dependent on amplification efficiency that can vary among targets and samples, accurate calculation of the target quantity and relative gene expression requires that the actual amplification efficiency be taken into account in the analysis and reports. PCR efficiency is frequently derived from standard curves, but this approach is affected by dilution errors and hampered by properties of the standard and the diluent. These factors affect accurate quantification of clinical and biological samples used in diagnostic applications and collected in challenging conditions. PCR efficiencies determined from individual amplification curves avoid these confounders. To obtain unbiased efficiency-corrected results, we recommend absolute quantification with a single undiluted calibrator with a known target concentration and efficiency values derived from the amplification curves of the calibrator and the unknown samples. SUMMARY: For meaningful diagnostics or biological interpretation, the reported results of qPCR experiments should be efficiency corrected. To avoid ambiguity, the Minimal Information for Publications on Quantitative Real-Time PCR Experiments (MIQE) guidelines checklist should be extended to require the methods that were used (1) to determine the PCR efficiency and (2) to calculate the reported target quantity and relative gene expression value.

Acute-onset high-morbidity primary photosensitisation in sheep associated with consumption of the Casbah and Mauro cultivars of the pasture legume Biserrula.

BACKGROUND: Primary photosensitisation (PS) subsequent to ingestion of the pasture legume Biserrula pelecinus L. (biserrula) has recently been confirmed in grazing livestock. Given the potential utility of this pasture species in challenging climates, a grazing trial was undertaken to examine if both varieties 'Casbah' and 'Mauro' were able to cause photosensitisation in livestock, and if this could be mitigated by grazing in winter, or in combination with other common pasture species. RESULTS: A controlled grazing trial was undertaken in winter in Australia with plots containing a dominant pasture of Biserrula pelecinus L. cv. 'Casbah' or 'Mauro', or mixed biserrula/perennial ryegrass populations. A photosensitisation grading system was established. 167 prime meat ewe lambs were introduced to the plots and monitored twice daily. Mild clinical signs were observed at 72 h on pasture. All animals were removed from biserrula dominant stands at this point. Four animals grazing 'Casbah' dominant pasture rapidly proceeded to severe photosensitisation in the following 12 h. Animals remaining on mixed biserrula/ryegrass stands did not exhibit severe PS but showed an 89% incidence of mild to moderate photosensitisation over the following 14 days. Animals on mixed lucerne showed significantly lower PS score than animals grazing biserrula varieties of any composition. The trial was halted at 14 days as only plots with low biserrula proportion still contained unaffected animals. Necropsy revealed severe multifocal erythematous ulcerations and alopecia of the ear pinnae, severe bilateral periorbital and conjunctival oedema and variably severe subcutaneous facial oedema. No evidence of hepatopathy was present. A diagnosis of acute unseasonal primary photosensitisation caused by biserrula ingestion with no other underlying pathology was confirmed. CONCLUSIONS: We report an unseasonal outbreak of acute photosensitisation in sheep grazing Biserrula pelecinus L cvs.'Casbah' and 'Mauro' with exceedingly high morbidity. A grading system is also proposed as a tool for objective and consistent clinical appraisal of future PS outbreaks. This finding expands our definition of seasonal and temporal risk periods for biserrula photosensitisation, and is the first to identify that both commercial cultivars of biserrula can cause primary photosensitisation in sheep.

Minicircle Mediated Gene Delivery to Canine and Equine Mesenchymal Stem Cells.

Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair.

Super pharmacological levels of calcitriol (1,25-(OH)2D3) inhibits mineral deposition and decreases cell proliferation in a strain dependent manner in chicken mesenchymal stem cells undergoing osteogenic differentiation in vitro.

The biologically active form of vitamin D3, calcitriol (1,25-(OH)2D3), plays a key role in mineral homeostasis and bone formation and dietary vitamin D3deficiency is a major cause of bone disorders in poultry. Supplementary dietary cholecalciferol (25-hydroxyvitamin D, 25-OH), the precursor of calcitriol, is commonly employed to combat this problem; however, dosage must be carefully determined as excess dietary vitamin D can cause toxicity resulting in a decrease in bone calcification, hypercalcinemia and renal failure. Despite much research on the therapeutic administration of dietary vitamin D in humans, the relative sensitivity of avian species to exogenous vitamin D has not been well defined. In order to determine the effects of exogenous 1,25-(OH)2D3during avian osteogenesis, chicken bone marrow-derived mesenchymal stem cells (BM-MSCs) were exposed to varying doses of 1,25-(OH)2D3during in vitro osteogenic differentiation and examined for markers of early proliferation and osteogenic induction. Similar to humans and other mammals, poultry BM-MSCs were found to be highly sensitive to exogenous 1,25-(OH)2D3with super pharmacological levels exerting significant inhibition of mineralization and loss of cell proliferation in vitro. Strain related differences were apparent, with BM-MCSs derived from layers strains showing a higher level of sensitivity to 1,25-(OH)2D3than those from broilers. These data suggest that understanding species and strain specific sensitivities to 1,25-(OH)2D3is important for optimizing bone health in the poultry industry and that use of avian BM-MSCs are a useful tool for examining underlying effects of genetic variation in poultry.

Secondary plant products causing photosensitization in grazing herbivores: their structure, activity and regulation.

Photosensitivity in animals is defined as a severe dermatitis that results from a heightened reactivity of skin cells and associated dermal tissues upon their exposure to sunlight, following ingestion or contact with UV reactive secondary plant products. Photosensitivity occurs in animal cells as a reaction that is mediated by a light absorbing molecule, specifically in this case a plant-produced metabolite that is heterocyclic or polyphenolic. In sensitive animals, this reaction is most severe in non-pigmented skin which has the least protection from UV or visible light exposure. Photosensitization in a biological system such as the epidermis is an oxidative or other chemical change in a molecule in response to light-induced excitation of endogenous or exogenously-delivered molecules within the tissue. Photo-oxidation can also occur in the plant itself, resulting in the generation of reactive oxygen species, free radical damage and eventual DNA degradation. Similar cellular changes occur in affected herbivores and are associated with an accumulation of photodynamic molecules in the affected dermal tissues or circulatory system of the herbivore. Recent advances in our ability to identify and detect secondary products at trace levels in the plant and surrounding environment, or in organisms that ingest plants, have provided additional evidence for the role of secondary metabolites in photosensitization of grazing herbivores. This review outlines the role of unique secondary products produced by higher plants in the animal photosensitization process, describes their chemistry and localization in the plant as well as impacts of the environment upon their production, discusses their direct and indirect effects on associated animal systems and presents several examples of well-characterized plant photosensitization in animal systems.

Meta-Analysis of qPCR for Bovine Respiratory Disease Based on MIQE Guidelines.

Qualitative and quantitative PCR-based tests are widely used in both diagnostics and research to assess the prevalence of disease-causing pathogens in veterinary medicine. The efficacy of these tests, usually measured in terms of sensitivity and specificity, is critical in confirming or excluding a clinical diagnosis. We undertook a meta-analysis to assess the inherent value of published PCR diagnostic approaches used to confirm and quantify bacteria and viruses associated with bovine respiratory disease (BRD) in cattle. This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A thorough search of nine electronic databases (Web of Science, EBSCOhost, Cambridge journals online, ProQuest, PubMed, Sage journals online, ScienceDirect, Wiley online library and MEDLINE) was undertaken to find studies that had reported on the use of PCR and/or qPCR for the detection and/or quantification of BRD associated organisms. All studies meeting the inclusion criteria for reporting quantitative PCR for identification of BRD associated microorganisms were included in the analysis. Studies were then assessed on the applications of the Minimum Information for Publication of Quantitative Real-Time PCR Experiment (MIQE) and PCR primer/probe sequences were extracted and tested for in silico specificity using a high level of stringency. Fourteen full-text articles were included in this study. Of these, 79% of the analysed articles did not report the application of the MIQE guidelines in their study. High stringency in silico testing of 144 previously published PCR primer/probe sequences found many to have questionable specificity. This review identified a high occurrence of primer/probe sequences with a variable in silico specificity such that this may have implications for the accuracy of reporting. Although this analysis was only applied to one specific disease state, identification of animals suspected to be suffering from bovine respiratory disease, there appears to be more broadly a need for veterinary diagnostic studies to adopt international best practice for reporting of quantitative PCR diagnostic data to be both accurate and comparable between studies and methodologies.

Producer practices and attitudes: Non-replacement male calf management in the Australian dairy industry.

Currently, there is no standardized rearing method or production guidelines for non-replacement male dairy calves that maximizes their economic viability. Producers have highlighted the need to match consumer expectations, but even with broadscale welfare improvement across the dairy industry, challenges remain at providing reliable and valuable pathways for non-replacement male dairy calves for beef production. A key consumer concern has been the use of on-farm euthanasia. Euthanasia has been a catalyst for change in the industry from a human and animal welfare perspective. The practice of euthanasia can lead to a decline in personnel wellbeing. To investigate the relationship between on-farm management practices of non-replacement male dairy calves and producer perceptions of their value proposition, an online questionnaire was provided to Australian dairy producers between June and October 2021. The aim was to identify supply-chain profitability of non-replacement male calves and investigate the attitudes and effects of euthanasia on producer wellbeing as part of managing these calves. A total of 127 useable responses were obtained, and a Bayesian network (BN) was utilized to model the interdependencies between management practices and wellbeing among participants. The results indicated that in general, dairy producers desired high welfare standards in their enterprises with regard to non-replacement male calves as well as expressed a desire to meet industry and consumers' expectations. In line with anecdotal reports of a reduction in practice, euthanasia was not identified as common practice in this group; however, producers were still accessing early-life markets for non-replacement male calves with operational requirements and environmental factors influencing their decisions. Producers expressed dissatisfaction with market access for their calves, as well as the lack of suitability of Australian beef grading standards for dairy-bred carcasses. Australian dairy managers and owners identified that euthanasia influenced employee wellbeing; however, they did not acknowledge euthanasia had an effect on their own wellbeing. Overall, the findings of this study indicate that all non-replacement male calf breeds had the potential to access profitable markets, and avoidance of euthanasia is a strong driver of change among dairy beef production systems in Australia.

Barriers to Utilizing Non-replacement Male Calves in the Australian Dairy Industry: A Qualitative Study.

Male non-replacement calves in dairy systems represent an underutilized economic resource for dairy producers worldwide. Despite this, increasing the practice of rearing non-replacement male calves has significant barriers both in on-farm adoption and practice. Poor neonatal rearing practices, higher levels of morbidity and mortality, and disaggregated production pathways with multiple points of handling, have all been described as barriers to adoption of surplus calf production. To identify the critical decision-determining challenges associated with broader adoption of raising non-replacement stock, and to investigate the whole-of-value chain issues faced by dairy producers to rear non-replacement male calves, we undertook a series of semi-structured interviews with Australian dairy producers to interrogate their key challenges. To achieve this, a constructivist grounded theory approach was used to inform the process of analysis of in-depth interviews with Australian dairy producers regarding their current practices and perceptions. Five major themes emerged from these conversations that were key barriers to on-farm non-replacement calf rearing in the producer group participants. These were: impacts of drought on cost and availability of feed for these calves and the whole herd; the management requirements of non-replacement male calves as an additional workload to that of their current operation; their attitudes and current practices to and surrounding euthanasia; perceived ease of supply-chain access for these calves, and their perceptions of the economic value of dairy-beef product as a return on investment. Understanding the barriers to adoption of non-replacement calf rearing, and addressing the value proposition for dairy beef, can assist increased uptake of non-replacement calf rearing. These findings will allow development of strategies to address these barriers, and extension of viable management strategies to increase adoption of profitable business practices surrounding non-replacement male calf production.

Genotypic identification of Panicum spp. in New South Wales, Australia using DNA barcoding.

Australia has over 30 Panicum spp. (panic grass) including several non-native species that cause crop and pasture loss and hepatogenous photosensitisation in livestock. It is critical to correctly identify them at the species level to facilitate the development of appropriate management strategies for efficacious control of Panicum grasses in crops, fallows and pastures. Currently, identification of Panicum spp. relies on morphological examination of the reproductive structures, but this approach is only useful for flowering specimens and requires significant taxonomic expertise. To overcome this limitation, we used multi-locus DNA barcoding for the identification of ten selected Panicum spp. found in Australia. With the exception of P. buncei, other native Australian Panicum were genetically separated at the species level and distinguished from non-native species. One nuclear (ITS) and two chloroplast regions (matK and trnL intron-trnF) were identified with varying facility for DNA barcode separation of the Panicum species. Concatenation of sequences from ITS, matK and trnL intron-trnF regions provided clear separation of eight regionally collected species, with a maximum intraspecific distance of 0.22% and minimum interspecific distance of 0.33%. Two of three non-native Panicum species exhibited a smaller genome size compared to native species evaluated, and we speculate that this may be associated with biological advantages impacting invasion of non-native Panicum species in novel locations. We conclude that multi-locus DNA barcoding, in combination with traditional taxonomic identification, provides an accurate and cost-effective adjunctive tool for further distinguishing Panicum spp. at the species level.

Gli3 is required autonomously for dorsal telencephalic cells to adopt appropriate fates during embryonic forebrain development.

The Gli3 zinc finger transcription factor is expressed in developing forebrain, with the highest levels of expression in dorsal telencephalon. In Gli3(-/-) embryos the dorsal telencephalon is abnormally small and fails to develop dorsomedial telencephalic structures, including hippocampus and cortical hem, while the ventral telencephalon appears to expand. A hurdle to understanding the underlying mechanisms is that abnormalities of developing Gli3(-/-) telencephalic cells in Gli3(-/-) mutants result from a combination of their own cell autonomous defects and defects in the Gli3(-/-) cells that surround them. Here we used chimeras to identify some of the defects of Gli3(-/-) telencephalic cells that are likely to be autonomous by studying how Gli3(-/-) cells develop when surrounded by a majority of wild-type cells. We found that Gli3(-/-) cells are present in all components of the Gli3(-/-)<-->Gli3(+/+) chimeric forebrain, including dorsomedial structures, in proportions that either equal or exceed proportions found elsewhere in the embryo. Gli3(-/-) cells segregate from Gli3(+/+) cells to form many abnormal structures particularly in dorsal telencephalon. Gli3(-/-) cells in some locations are misspecified: in those parts of the dorsal telencephalon near to its boundaries with the diencephalon and the ventral telencephalon, mutant cells express sets of transcription factors expressed by wild-type cells on the other side of the boundary. Elsewhere in the dorsal telencephalon, in the diencephalon and in the ventral telencephalon, mutant cells express sets of transcription factors similar to those expressed by their immediately surrounding wild-type cells. We propose that an important cell autonomous action of Gli3 is to regulate the competence of dorsal telencephalic cells, preventing cells near to its boundaries expressing regulatory factors normally restricted to adjacent tissues.

Novel lines of Pax6-/- embryonic stem cells exhibit reduced neurogenic capacity without loss of viability.

BACKGROUND: Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells died rapidly after neuronal differentiation in vitro. RESULTS: Here we report the derivation of new lines of Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- <--> Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- <--> Pax6+/+ chimeras made by embryo aggregation. CONCLUSIONS: We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development.

PGRMC1 effects on metabolism, genomic mutation and CpG methylation imply crucial roles in animal biology and disease.

BACKGROUND: Progesterone receptor membrane component 1 (PGRMC1) is often elevated in cancers, and exists in alternative states of phosphorylation. A motif centered on PGRMC1 Y180 was evolutionarily acquired concurrently with the embryological gastrulation organizer that orchestrates vertebrate tissue differentiation. RESULTS: Here, we show that mutagenic manipulation of PGRMC1 phosphorylation alters cell metabolism, genomic stability, and CpG methylation. Each of several mutants elicited distinct patterns of genomic CpG methylation. Mutation of S57A/Y180/S181A led to increased net hypermethylation, reminiscent of embryonic stem cells. Pathways enrichment analysis suggested modulation of processes related to animal cell differentiation status and tissue identity, as well as cell cycle control and ATM/ATR DNA damage repair regulation. We detected different genomic mutation rates in culture. CONCLUSIONS: A companion manuscript shows that these cell states dramatically affect protein abundances, cell and mitochondrial morphology, and glycolytic metabolism. We propose that PGRMC1 phosphorylation status modulates cellular plasticity mechanisms relevant to early embryological tissue differentiation.

The aetiology, prevalence and morbidity of outbreaks of photosensitisation in livestock: A review.

BACKGROUND: Photosensitisation is a clinical condition occurring in both humans and animals that causes significant injury to affected individuals. In livestock, outbreaks of photosensitisation caused by ingestion of toxic plants are relatively common and can be associated with significant economic loss. OBJECTIVES: The agents that are most commonly implicated in outbreaks of photosensitisation have not been formally investigated on a global scale. To address this question, a systematic review of the literature was undertaken to determine the most common causative agents implicated in outbreaks of photosensitisation in livestock in Australia and globally, as well as the prevalence and morbidity of such outbreaks. METHODS: A systematic database search was conducted to identify peer-reviewed case reports of photosensitisation in livestock published worldwide between 1900 and April 2018. Only case reports with a full abstract in English were included. Non peer-reviewed reports from Australia were also investigated. Case reports were then sorted by plant and animal species, type of photosensitisation by diagnosis, location, morbidity and mortality rate and tabulated for further analysis. RESULTS: One hundred and sixty-six reports qualified for inclusion in this study. Outbreaks were reported in 20 countries. Australia (20), Brazil (20) and the United States (11) showed the highest number of peer-reviewed photosensitisation case reports from this analysis. Hepatogenous (Type III) photosensitisation was the most frequently reported diagnosis (68.5%) and resulted in higher morbidity. Panicum spp., Brachiaria spp. and Tribulus terrestris were identified as the most common causes of hepatogenous photosensitisation globally. CONCLUSIONS: Hepatogenous photosensitisation in livestock represents a significant risk to livestock production, particularly in Australia, Brazil, and the United States. Management of toxic pastures and common pasture weeds may reduce the economic impact of photosensitisation both at a national and global level.

Chemometric analysis of Amaranthus retroflexus in relation to livestock toxicity in southern Australia.

Amaranthus retroflexus L., an introduced invasive weed in southern Australia, has been associated with acute renal failure and/or mortality in a number of livestock species. While its leaves, flowers and stems are generally reported to contain high levels of nitrogen, few studies have fully characterised the chemical composition of A. retroflexus foliage with respect to mammalian toxicity. We performed extensive metabolic profiling of stems, leaves, roots and inflorescence tissues of A. retroflexus collected from three spatially and/or temporally distinct toxicity outbreaks, and report on the 1) composition of primary and secondary metabolites in methanolic extracts of A. retroflexus tissues using HPLC and HPLC-MS QToF and 2) chemometric analysis of A. retroflexus extracts in relation to the associated toxin(s). All tissues of A. retroflexus possessed an abundance of N-containing metabolites, particularly quaternary ammonium compounds which were identified as betaines, two of which (valine betaine and isoleucine betaine) are rarely encountered in plants. Cytotoxicity to murine fibroblasts was highest in extracts of leaf tissue and was associated with a single, a small modified peptide with high similarity to N-acetyl-L-α-aspartyl-L-alanyl-L-α-aspartyl-L-α-glutamyl-O-(carboxymethyl)-L-tyrosyl-L-leucinamide, a synthetic phosphotyrosyl mimic involved in cell signaling processes. One possible mode of action leading to acute renal failure in grazing livestock by a modified peptide such as this is proposed.

Mouse models of telencephalic development.

As the telencephalon first emerges from anterior neuroectoderm, signalling molecules and transcription factors combine to specify the identity and fate of cells in each of its regions. Studies of both naturally occurring and transgenic mutant mice have identified many genes that contribute to this process. The development of telencephalon and its regions is specified by signalling molecules produced at sites both surrounding and within the telencephalon. Different parts of the telencephalon express different combinations of transcription factors that control processes including proliferation, cell fate determination and migration in order to create the unique phenotype of each region.

STEROIDAL SAPONIN TOXICITY IN EASTERN GREY KANGAROOS ( MACROPUS GIGANTEUS): A NOVEL CLINICOPATHOLOGIC PRESENTATION OF HEPATOGENOUS PHOTOSENSITIZATION.

We describe the clinicopathologic features of a mortality event characterized by blindness and dermatitis affecting eastern grey kangaroos ( Macropus giganteus), secondary to hepatogenous photosensitization. Affected animals exhibited photophobic behavior, blindness, ataxia, recumbency, lethargy, ear shaking, and behavior consistent with distress or depression. The photophobia manifested as abnormal shade-seeking during the day, including finding refuge under or in structures used frequently by people. Severely affected kangaroos were jaundiced and had markedly elevated serum bilirubin and gamma glutamyl-transpeptidase concentrations. Blindness in affected animals was attributed to moderate to severe corneal opacity due to corneal edema and inflammation. Skin lesions were typically subtle on gross examination even in cases which had severe necrotizing dermatitis histologically. Histologic lesions in the liver of affected animals included the presence of acicular clefts typical of steroidal saponins. The outbreak was associated with pasture dominated by the invasive grass, Panicum gilvum, which is a recognized source of saponin-induced photosensitization in livestock.

Penetrance of eye defects in mice heterozygous for mutation of Gli3 is enhanced by heterozygous mutation of Pax6.

BACKGROUND: Knowledge of the consequences of heterozygous mutations of developmentally important genes is important for understanding human genetic disorders. The Gli3 gene encodes a zinc finger transcription factor and homozygous loss-of-function mutations of Gli3 are lethal. Humans heterozygous for mutations in this gene suffer Greig cephalopolysyndactyly or Pallister-Hall syndromes, in which limb defects are prominent, and mice heterozygous for similar mutations have extra digits. Here we examined whether eye development, which is abnormal in mice lacking functional Gli3, is defective in Gli3+/- mice. RESULTS: We showed that Gli3 is expressed in the developing eye but that Gli3+/- mice have only very subtle eye defects. We then generated mice compound heterozygous for mutations in both Gli3 and Pax6, which encodes another developmentally important transcription factor known to be crucial for eye development. Pax6+/-; Gli3+/- eyes were compared to the eyes of wild-type, Pax6+/- or Gli3+/- siblings. They exhibited a range of abnormalities of the retina, iris, lens and cornea that was more extensive than in single Gli3+/- or Pax6+/- mutants or than would be predicted by addition of their phenotypes. CONCLUSION: These findings indicate that heterozygous mutations of Gli3 can impact on eye development. The importance of a normal Gli3 gene dosage becomes greater in the absence of a normal Pax6 gene dosage, suggesting that the two genes co-operate during eye morphogenesis.

Disruption of early events in thalamocortical tract formation in mice lacking the transcription factors Pax6 or Foxg1.

Early events in the formation of the thalamocortical tract remain poorly understood. Recent work has suggested that thalamocortical axons follow a path pioneered by transient thalamic afferents originating from the medial part of the ventral telencephalon. We studied the development of these transient afferents and the thalamocortical tract in mutant mice lacking transcription factors normally expressed in the dorsal thalamus or ventral telencephalon. Pax6 is expressed in the dorsal thalamus, but not in the medial part of the ventral telencephalon, and the thalamocortical tract fails to form in Pax6(-/-) embryos. We found that transient thalamic afferents from the ventral telencephalon do not form in Pax6(-/-) embryos; this may contribute to the failure of their thalamocortical development. The distribution of Pax6(-/-) cells in Pax6(-/-)<--> Pax6(+/+) chimeras supports conclusions drawn from forebrain marker gene expression that Pax6 is not required for the normal development of the medial part of the ventral telencephalon but is required in the dorsal thalamus. Failure of the transient afferent pathway to develop is therefore likely a cell nonautonomous defect reflecting primary defects in the thalamus. We then examined the formation of thalamic afferents and efferents in Foxg1(-/-) embryos, which lack recognizable ventral telencephalic structures. In these embryos thalamic efferents navigate correctly through the thalamus but fail to turn laterally into the telencephalon, whereas other axons are able to cross the diencephalic/telencephalic boundary. Our results support a role for the ventral telencephalon in guiding the early development of the thalamocortical tract and identify a new role for the transcription factor Pax6 in regulating the ability of the thalamus to attract ventral telencephalic afferents.

Complex Membrane Channel Blockade: A Unifying Hypothesis for the Prodromal and Acute Neuropsychiatric Sequelae Resulting from Exposure to the Antimalarial Drug Mefloquine.

The alkaloid toxin quinine and its derivative compounds have been used for many centuries as effective medications for the prevention and treatment of malaria. More recently, synthetic derivatives, such as the quinoline derivative mefloquine (bis(trifluoromethyl)-(2-piperidyl)-4-quinolinemethanol), have been widely used to combat disease caused by chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum. However, the parent compound quinine, as well as its more recent counterparts, suffers from an incidence of adverse neuropsychiatric side effects ranging from mild mood disturbances and anxiety to hallucinations, seizures, and psychosis. This review considers how the pharmacology, cellular neurobiology, and membrane channel kinetics of mefloquine could lead to the significant and sometimes life-threatening neurotoxicity associated with mefloquine exposure. A key role for mefloquine blockade of ATP-sensitive potassium channels and connexins in the substantia nigra is considered as a unifying hypothesis for the pathogenesis of severe neuropsychiatric events after mefloquine exposure in humans.