Meiocyte size is a determining factor for unreduced gamete formation in Arabidopsis thaliana.

Polyploidy, the presence of more than two sets of chromosomes within a cell, is a widespread phenomenon in plants. The main route to polyploidy is considered through the production of unreduced gametes that are formed as a consequence of meiotic defects. Nevertheless, for reasons poorly understood, the frequency of unreduced gamete formation differs substantially among different plant species. The previously identified meiotic mutant jason (jas) in Arabidopsis thaliana forms about 60% diploid (2n) pollen. JAS is required to maintain an organelle band as a physical barrier between the two meiotic spindles, preventing previously separated chromosome groups from uniting into a single cell. In this study, we characterized the jas suppressor mutant telamon (tel) that restored the production of haploid pollen in the jas background. The tel mutant did not restore the organelle band, but enlarged the size of male jas tel meiocytes, suggesting that enlarged meiocytes can bypass the requirement of the organelle band. Consistently, enlarged meiocytes generated by a tetraploid jas mutant formed reduced gametes. The results reveal that meiocyte size impacts chromosome segregation in meiosis II, suggesting an alternative way to maintain the ploidy stability in meiosis during evolution.

MRI diagnosis of spontaneous intraventricular tension-pneumocephalus in a 10-month-old male Saarloos Wolfdog.

A 10-month-old male Saarloos Wolfdog was presented with a history of multiple neurologic deficits that had acutely progressed. Neurologic examination findings localized signs to the cerebrum and brainstem. Magnetic resonance imaging revealed markedly enlarged and gas-filled lateral ventricles with a mass effect leading to cerebellar herniation. A right-sided defect of the cribriform plate with a dysplastic ethmoturbinate was identified as the inlet of air and origin of the intraventricular tension pneumocephalus. Surgical findings were consistent with a ruptured, congenital, nasal meningocele.

Postzygotic reproductive isolation established in the endosperm: mechanisms, drivers and relevance.

The endosperm is a developmental innovation of angiosperms that supports embryo growth and germination. Aside from this essential reproductive function, the endosperm fuels angiosperm evolution by rapidly establishing reproductive barriers between incipient species. Specifically, the endosperm prevents hybridization of newly formed polyploids with their non-polyploid progenitors, a phenomenon termed the triploid block. Furthermore, recently diverged diploid species are frequently reproductively isolated by endosperm-based hybridization barriers. Current genetic approaches have revealed a prominent role for epigenetic processes establishing these barriers. In particular, imprinted genes, which are expressed in a parent-of-origin-specific manner, underpin the interploidy barrier in the model species Arabidopsis. We will discuss the mechanisms establishing hybridization barriers in the endosperm, the driving forces for these barriers and their impact for angiosperm evolution. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

On the origin of the widespread self-compatible allotetraploid Capsella bursa-pastoris (Brassicaceae).

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed ~300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.

The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons.

MADS-box transcription factors (TFs) are ubiquitous in eukaryotic organisms and play major roles during plant development. Nevertheless, their function in seed development remains largely unknown. Here, we show that the imprinted Arabidopsis thaliana MADS-box TF PHERES1 (PHE1) is a master regulator of paternally expressed imprinted genes, as well as of non-imprinted key regulators of endosperm development. PHE1 binding sites show distinct epigenetic modifications on maternal and paternal alleles, correlating with parental-specific transcriptional activity. Importantly, we show that the CArG-box-like DNA-binding motifs that are bound by PHE1 have been distributed by RC/Helitron transposable elements. Our data provide an example of the molecular domestication of these elements which, by distributing PHE1 binding sites throughout the genome, have facilitated the recruitment of crucial endosperm regulators into a single transcriptional network.

[Evaluation of the accuracy of volume navigation of sonography and computed tomography using a phantom]. / Genauigkeitsevaluierung der Volumennavigation von Sonografie und Computertomografie anhand eines Phantoms.

BACKGROUND AND OBJECTIVE: In case of superimpositions of gas in the gastrointestinal tract or the ribs, tissue changes well detectable on computed tomography (CT) cannot be identified sonographically in a number of cases. Combining ultrasonography and CT provides enhanced information compared to sole sonography and volume navigation may be used as an effective tool. Tissue samples easily and safely obtained under sonographic guidance are often necessary to confirm the diagnosis of a suspicious focus. In these cases, the spatial fusion of CT and sonography may also be employed for improved visualization of foci by eliminating superimposition of sonographic images which is a limitation of ultrasound. This study investigated the potential benefit and improved informative value of the fusion of CT and sonography in case of superimpositions and aimed at determining the registration method with the best accuracy. MATERIALS AND METHODS: Sixteen models (10 models with peas [low contrast], 6 models with wooden spheres [high contrast] as round structures) were created. These models were examined by computed tomography and fused using 3 volume navigation protocols. Subsequently, volume-guided sonography was performed. The deviation of the specimens was measured. RESULTS: In total, 1026 measurements of the pea models and 648 measurements of the wooden sphere models were carried out. A fusion accuracy of 100 % was observed in 9.9 % (102/1026) resp. 9.9 % (64/648) of the models. In 85.4 % (876/1026) resp. 94.1 % (610/648) the deviation was < 5 mm and in 98.1 % (1006/1026) resp. 99.4 % (644/648) it was < 10 mm. The registration protocol in which all reference points were used for spatial fusion proved to be the most accurate CONCLUSION: The registration protocols for volume-guided ultrasound have sufficient biopsy accuracy to merge identical sites and provide the basis for improved volume-navigated biopsy sampling.

[Contrast-enhanced ultrasound at a rare bilateral renal carcinoma with secondary inflammation and necrosis in a cat]. / Kontrastmittelsonografie bei einem seltenen bilateralen renalen Karzinom mit sekundärer Entzündung und Nekrose bei einer Katze.

A 17-year-old male neutered domestic short hair cat was presented because of anorexia. The clinical examination revealed no abnormalities. Using sonography, mainly hypoechoic mass lesions at the level of the cortex and capsule were detected in both kidneys. The severity of the renal lesions could be clearly demonstrated using contrast-enhanced ultrasound and contrast-enhanced computed tomography. Under general anesthesia, fine needle aspirations of the lesions were taken. Part of the lesions were sampled from dorsal, an unusual practice for small animal medicine. Cytology revealed a bilateral renal carcinoma with secondary inflammation and necrosis. The cat improved under medical symptomatic treatment, but was euthanized 2 weeks later.

Needle-based storage-phosphor detector radiography is superior to a conventional powder-based storage phosphor detector and a high-resolution screen-film system in small patients (budgerigars and mice).

This method comparison study used radiographs of 20 mice and 20 budgerigars to investigate comparability between computed radiography (CR) and high-resolution screen-film systems and study the effects of reduced radiation doses on image quality of digital radiographs of small patients. Exposure settings used with the mammography screen-film system (SF) were taken as baseline settings. A powder-based storage-phosphor system (CRP) and a needle-based storage-phosphor system (CRN) were used with the same settings (D/100%) and half the detector dose (D/50%). Using a scoring system four reviewers assessed five criteria per species covering soft tissue and bone structures. Results were evaluated for differences between reviewers (interobserver variability), systems and settings (intersystem variability, using visual grading characteristic analysis). Correlations were significant (p ≤ 0.05) for interobserver variability in 86.7% of the cases. Correlation coefficients ranged from 0.206 to 0.772. For mice and budgerigars, the CRN system was rated as superior to the SF and CRP system for most criteria, being significant in two cases each. Comparing the SF and CRP system, the conventional method scored higher for all criteria, in one case significantly. For both species and both digital systems, dose reduction to 50% resulted in significantly worse scores for most criteria. In summary, the needle-based storage-phosphor technique proved to be superior compared to the conventional storage-phosphor and mammography screen-film system. Needle-based detector systems are suitable substitutes for high-resolution screen-film systems when performing diagnostic imaging of small patients. Dose reduction to 50% of the corresponding dose needed in high-resolution film-screen systems cannot be recommended.

Auxin regulates endosperm cellularization in Arabidopsis.

The endosperm is an ephemeral tissue that nourishes the developing embryo, similar to the placenta in mammals. In most angiosperms, endosperm development starts as a syncytium, in which nuclear divisions are not followed by cytokinesis. The timing of endosperm cellularization largely varies between species, and the event triggering this transition remains unknown. Here we show that increased auxin biosynthesis in the endosperm prevents its cellularization, leading to seed arrest. Auxin-overproducing seeds phenocopy paternal-excess triploid seeds derived from hybridizations of diploid maternal plants with tetraploid fathers. Concurrently, auxin-related genes are strongly overexpressed in triploid seeds, correlating with increased auxin activity. Reducing auxin biosynthesis and signaling reestablishes endosperm cellularization in triploid seeds and restores their viability, highlighting a causal role of increased auxin in preventing endosperm cellularization. We propose that auxin determines the time of endosperm cellularization, and thereby uncovered a central role of auxin in establishing hybridization barriers in plants.

[Nephroblastoma in a 2-year-old female dog]. / Nephroblastom bei einer 2-jährigen Hündin.

An intact female 2-year-old boxer presented with polydipsia, polyuria, and decreased feed intake. Palpation of the abdomen was painful. Sonography revealed an abdominal mass of the left kidney causing displacement of the organs located in the cranial and mid-abdomen. Dimen sion and invasiveness of the process were evaluated both by contrast enhanced ultrasound (CEUS) and contrast-enhanced computed tomography. Histopathological examination of a biopsy sample revealed a nephroblastoma. The case report describes the clinical, sonographic, and computed tomographic results and the outcome in the untreated dog over a period of 5 months.

[Multiple choristoma and a myelolipoma in a Sheltie]. / Multiple Choristome und ein Myelolipom bei einem Sheltie.

An 11-year-old female Sheltie was presented with inappetence and a progressive increase in abdominal distention. Abdominal ultrasound revealed a large cystic mass in the midabdomen and cystic lesions in the right liver lobe and in the caudal pole of the left kidney. Histopathologic examination of the resected tissue revealed a myelolipoma of the spleen, dispersed splenic tissue in the liver and dispersed uterine and salpinx tissues in the kidney. This report describes the clinical, ultrasonographic and computed tomographic features and the results of histopathology. In addition to the abnormally large and cystic myelolipoma of the spleen, the great number of choristomas is remarkable, which has not previously been documented in a dog.

[Complications of ultrasound-guided liver biopsies in dogs and cats]. / Komplikationen sonographisch gestützter Leberbiopsien bei Hund und Katze.

OBJECTIVE: Ultrasound-guided biopsies are obtained generally to reach a sound diagnosis in the case of sonographically detectable hepatic changes. The most common and well-known complication associated with ultrasound-guided biopsies is haemorrhage after sampling, which may lead to the patient's death. The aim of this study was to evaluate a possible association between alterations in the coagulation parameters and sonographically observable haemorrhage following liver biopsies. MATERIALS AND METHODS: Dogs and cats in which an ultrasound-guided biopsy of the liver had been performed were retrospectively enrolled in the study. Inclusion criteria were thoroughly documented sonographic findings and the results of the coagula tion parameter thromboplastin time or a combination of the activated partial thromboplastin time and prothrombin time. The incidence of post-interventional ascites and possible risk factors were evaluated. RESULTS: A total of 105 patients met the inclusion criteria and the data of 94 dogs (89.5 %) and 11 cats (10.5 %) were analysed. Post-interventional complications occurred in 26 of 105 patients (24.8 %), 21.9 % being minor and 2.9 % being major. Free abdominal fluid after intervention was sonographically detected in 22 of 94 dogs (23.4 %) and four of 11 cats (36.4 %). Three of 10 dogs (2.9 %) with a clinically significant prolongated coagulation time of > 25 % had ascites after biopsy. Only a small amount of fluid was detected in two of these three dogs, while a moderate amount of ascites was observed in the third dog. One of two cats with a prolonged coagulation time of > 25 % developed a moderate amount of ascites after the puncture. No statistically significant association was found between the occurrence of post-interventional ascites and a prolongation of the coagulation time. CONCLUSION: According to the study results, there is no obvi ous correlation between alterations in coagulation and haemorrhage after an ultrasound-guided liver biopsy.

Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis.

Chromosome dosage has substantial effects on reproductive isolation and speciation in both plants and animals, but the underlying mechanisms are largely obscure 1 . Transposable elements in animals can regulate hybridity through maternal small RNA 2 , whereas small RNAs in plants have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of long terminal repeat (LTR) retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21-22-nucleotide (nt) small RNAs in a dose-dependent fashion via RNA polymerase IV. We show that these epigenetically activated small interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (the 'triploid block'), and that natural variation for miR845 may account for 'endosperm balance' allowing the formation of triploid seeds. Targeting of the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.

Ectopic application of the repressive histone modification H3K9me2 establishes post-zygotic reproductive isolation in Arabidopsis thaliana.

Hybrid seed lethality as a consequence of interspecies or interploidy hybridizations is a major mechanism of reproductive isolation in plants. This mechanism is manifested in the endosperm, a dosage-sensitive tissue supporting embryo growth. Deregulated expression of imprinted genes such as ADMETOS (ADM) underpin the interploidy hybridization barrier in Arabidopsis thaliana; however, the mechanisms of their action remained unknown. In this study, we show that ADM interacts with the AT hook domain protein AHL10 and the SET domain-containing SU(VAR)3-9 homolog SUVH9 and ectopically recruits the heterochromatic mark H3K9me2 to AT-rich transposable elements (TEs), causing deregulated expression of neighboring genes. Several hybrid incompatibility genes identified in Drosophila encode for dosage-sensitive heterochromatin-interacting proteins, which has led to the suggestion that hybrid incompatibilities evolve as a consequence of interspecies divergence of selfish DNA elements and their regulation. Our data show that imbalance of dosage-sensitive chromatin regulators underpins the barrier to interploidy hybridization in Arabidopsis, suggesting that reproductive isolation as a consequence of epigenetic regulation of TEs is a conserved feature in animals and plants.

H3K36ac Is an Evolutionary Conserved Plant Histone Modification That Marks Active Genes.

In eukaryotic cells, histones are subject to a large number of posttranslational modifications whose sequential or combinatorial action affects chromatin structure and genome function. We identified acetylation at Lys-36 in histone H3 (H3K36ac) as a new chromatin modification in plants. The H3K36ac modification is evolutionary conserved in seed plants, including the gymnosperm Norway spruce (Picea abies) and the angiosperms rice (Oryza sativa), tobacco (Nicotiana tabacum), and Arabidopsis (Arabidopsis thaliana). In Arabidopsis, H3K36ac is highly enriched in euchromatin but not in heterochromatin. Genome-wide chromatin immunoprecipitation sequencing experiments revealed that H3K36ac peaks at the 5' end of genes, mainly on the two nucleosomes immediately distal to the transcription start site, independently of gene length. H3K36ac overlaps with H3K4me3 and the H2A.Z histone variant. The histone acetyl transferase GCN5 and the histone deacetylase HDA19 are required for H3K36ac homeostasis. H3K36ac and H3K36me3 show negative crosstalk, which is mediated by GCN5 and the histone methyl transferase SDG8. Although H3K36ac is associated with gene activity, we did not find a linear relationship between H3K36ac and transcript levels, suggesting that H3K36ac is a binary indicator of transcription.

SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content.

Key message: Purification of pollen nuclei. Germ cell epigenetics is a critical topic in plants and animals. The male gametophyte (pollen) of flowering plants is an attractive model to study genetic and epigenetic reprogramming during sexual reproduction, being composed of only two sperm cells contained within, its companion, vegetative cell. Here, we describe a simple and efficient method to purify SYBR Green-stained sperm and vegetative cell nuclei of Arabidopsis thaliana pollen using fluorescence-activated cell sorting to analyze chromatin and RNA profiles. The method obviates generating transgenic lines expressing cell-type-specific fluorescence reporters and facilitates functional genomic analysis of various mutant lines and accessions. We evaluate the purity and quality of the sorted pollen nuclei and analyze the technique's molecular basis. Our results show that both DNA and RNA contents contribute to SYBR Green-activated nucleus sorting and RNA content differences impact on the separation of sperm and vegetative cell nuclei. We demonstrate the power of the approach by sorting wild-type and polyploid mutant sperm and vegetative cell nuclei from mitotic and meiotic mutants, which is not feasible using cell-type-specific transgenic reporters. Our approach should be applicable to pollen nuclei of crop plants and possibly to cell/nucleus types and cell cycle phases of different species containing substantially different amounts of DNA and/or RNA.

Hypomethylated pollen bypasses the interploidy hybridization barrier in Arabidopsis.

Plants of different ploidy levels are separated by a strong postzygotic hybridization barrier that is established in the endosperm. Deregulated parent-of-origin specific genes cause the response to interploidy hybridizations, revealing an epigenetic basis of this phenomenon. In this study, we present evidence that paternal hypomethylation can bypass the interploidy hybridization barrier by alleviating the requirement for the Polycomb Repressive Complex 2 (PRC2) in the endosperm. PRC2 epigenetically regulates gene expression by applying methylation marks on histone H3. Bypass of the barrier is mediated by suppressed expression of imprinted genes. We show that the hypomethylated pollen genome causes de novo CHG methylation directed to FIS-PRC2 target genes, suggesting that different epigenetic modifications can functionally substitute for each other. Our work presents a method for the generation of viable triploids, providing an impressive example of the potential of epigenome manipulations for plant breeding.

An imprinted gene underlies postzygotic reproductive isolation in Arabidopsis thaliana.

Postzygotic reproductive isolation in response to interploidy hybridizations is a well-known phenomenon in plants that forms a major path for sympatric speciation. A main determinant for the failure of interploidy hybridizations is the endosperm, a nutritious tissue supporting embryo growth, similar to the functional role of the placenta in mammals. Although it has been suggested that deregulated imprinted genes underpin dosage sensitivity of the endosperm, the molecular basis for this phenomenon remained unknown. In a genetic screen for suppressors of triploid seed abortion, we have identified the paternally expressed imprinted gene ADMETOS (ADM). Here, we present evidence that increased dosage of ADM causes triploid seed arrest. A large body of theoretical work predicted that deregulated imprinted genes establish the barrier to interploidy hybridization. Our study thus provides evidence strongly supporting this hypothesis and generates the molecular basis for our understanding of postzygotic hybridization barriers in plants.

Increased maternal genome dosage bypasses the requirement of the FIS polycomb repressive complex 2 in Arabidopsis seed development.

Seed development in flowering plants is initiated after a double fertilization event with two sperm cells fertilizing two female gametes, the egg cell and the central cell, leading to the formation of embryo and endosperm, respectively. In most species the endosperm is a polyploid tissue inheriting two maternal genomes and one paternal genome. As a consequence of this particular genomic configuration the endosperm is a dosage sensitive tissue, and changes in the ratio of maternal to paternal contributions strongly impact on endosperm development. The fertilization independent seed (FIS) Polycomb Repressive Complex 2 (PRC2) is essential for endosperm development; however, the underlying forces that led to the evolution of the FIS-PRC2 remained unknown. Here, we show that the functional requirement of the FIS-PRC2 can be bypassed by increasing the ratio of maternal to paternal genomes in the endosperm, suggesting that the main functional requirement of the FIS-PRC2 is to balance parental genome contributions and to reduce genetic conflict. We furthermore reveal that the AGAMOUS LIKE (AGL) gene AGL62 acts as a dosage-sensitive seed size regulator and that reduced expression of AGL62 might be responsible for reduced size of seeds with increased maternal genome dosage.