Mesencephalic Astrocyte-Derived Neurotrophic Factor Regulates Morphology of Pigment-Dispersing Factor-Positive Clock Neurons and Circadian Neuronal Plasticity in Drosophila melanogaster.

Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is one of a few neurotrophic factors described in Drosophila melanogaster (DmMANF) but its function is still poorly characterized. In the present study we found that DmMANF is expressed in different clusters of clock neurons. In particular, the PDF-positive large (l-LNv) and small (s-LNv) ventral lateral neurons, the CRYPTOCHROME-positive dorsal lateral neurons (LNd), the group 1 dorsal neurons posterior (DN1p) and different tim-positive cells in the fly's visual system. Importantly, DmMANF expression in the ventral lateral neurons is not controlled by the clock nor it affects its molecular mechanism. However, silencing DmMANF expression in clock neurons affects the rhythm of locomotor activity in light:dark and constant darkness conditions. Such phenotypes correlate with abnormal morphology of the dorsal projections of the s-LNv and with reduced arborizations of the l-LNv in the medulla of the optic lobe. Additionally, we show that DmMANF is important for normal morphology of the L2 interneurons in the visual system and for the circadian rhythm in the topology of their dendritic tree. Our results indicate that DmMANF is important not only for the development of neurites but also for maintaining circadian plasticity of neurons.

The effectiveness of fibrolytic enzymes and active yeast on improving reticulorumen pH in dairy cows.

Exogenous fibrolytic enzymes (EFE) and yeast are feed supplements that improve forage digestion in rumen, but their influences on physical reticulorumen parameters are not well studied. This study was designed to evaluate the effect of the EFE:endo-ß-xylanase (37x104 U/cow/day), endocellulase (45x104 U/cow/day), endo-ß-glucanase (12x104U/cow/day), and active yeast - Saccharomyces cerevisiae CNCM-1077 (10x109CFU/cow/day) supplements on reticulorumen pH (RpH) and temperature (RT) in dairy cows. Nine Lithuanian Red cows were allocated into three groups (3 cows/group): control group (C) - farm diet without supplementation, enzyme group (E) - farm diet supplemented with EFE, enzyme and active yeast group (EY) - farm diet supplemented with EFE and active yeast. The feeding trial lasted for 60 d. All cows were equipped with reticuloruminal telemetric pH and temperature sensor device. Data provided by the device were used to calculate the mean RpH (RpH/24h), the mean minimal RpH (minRpH/24h) and mean of the time that RpH was below the threshold value of 6.0 (RpH⟨6.0/24h, min.). The highest RpH/24h (6.37±0.22) was observed in group EY and it was by 1.62% (p⟨0.05) and 1.27% (p⟨0.001) higher as compared with groups E and C, respectively. Also minRpH/24h (6.24±0.24) was highest in group EY and values were by 0.63% (p⟨0.001) and 0.65% (p⟨0.001) higher as compared with groups C and E, respectively. The shortest duration of RpH⟨6.0/24h, was recorded in group EY, and it was by 57.76% (p⟨0.05) and 47.87% shorter as compared with groups C and E, respectively. In conclusion, feed supplementation with EFE and Saccharomyces cerevisiae CNCM-1077 had beneficial effect on RpH.

PWP1 promotes nutrient-responsive expression of 5S ribosomal RNA.

PWP1 is a chromatin binding protein with an important role in animal growth control downstream of mTOR-mediated nutrient sensing. PWP1 has been shown to control tissue growth by promoting the transcription of 5.8S, 18S and 28S ribosomal RNAs (rRNAs) by RNA polymerase I (Pol I). Concomitantly with Pol I, RNA Polymerase III (Pol III) contributes to ribosome biogenesis by transcribing 5S rRNA in the nucleoplasm. Pol III activity is also closely controlled by nutrient-dependent signaling, however, how the activities of Pol I and Pol III are coordinated in response to nutrient-derived signals remains insufficiently understood. Experiments in Drosophila larvae and human cells reported here show that PWP1 associates with the chromatin at the 5S rDNA loci and is needed for nutrient-induced expression of 5S rRNA. Similar to the Pol I target rDNAs, PWP1 epigenetically maintains 5S rDNA in a transcription competent state. Thus, as a common regulator of Pol I and Pol III, PWP1 might contribute to coordinated control of ribosomal gene expression in response to nutrition.This article has an associated First Person interview with the first author of the paper.

Plant oil supplements reduce methane emissions and improve milk fatty acid composition in dairy cows fed grass silage-based diets without affecting milk yield.

Four lipid supplements varying in chain length or degree of unsaturation were examined for their effects on milk yield and composition, ruminal CH4 emissions, rumen fermentation, nutrient utilization, and microbial ecology in lactating dairy cows. Five Nordic Red cows fitted with rumen cannulas were used in a 5 × 5 Latin square with five 28-d periods. Treatments comprised total mixed rations based on grass silage with a forage-to-concentrate ratio of 60:40 supplemented with no lipid (CO) or 50 g/kg of diet dry matter (DM) of myristic acid (MA), rapeseed oil (RO), safflower oil (SO), or linseed oil (LO). Feeding MA resulted in the lowest DM intake, and feeding RO reduced DM intake compared with CO. Feeding MA reduced the yields of milk, milk constituents, and energy-corrected milk. Plant oils did not influence yields of milk and milk constituents, but reduced milk protein content compared with CO. Treatments had no effect on rumen fermentation characteristics, other than an increase in ammonia-N concentration due to feeding MA, RO, and SO compared with CO. Lipid supplements reduced daily ruminal CH4 emission; however, the response was to some extent a result of lower feed intake. Lipids modified microbial community structure without affecting total counts of bacteria, archaea, and ciliate protozoa. Dietary treatments had no effect on the apparent total tract digestibility of organic matter, fiber, and gross energy. Treatments did not affect either energy secreted in milk as a proportion of energy intake or efficiency of dietary N utilization. All lipids lowered de novo fatty acid synthesis in the mammary gland. Plant oils increased proportions of milk fat 18:0, cis 18:1, trans and monounsaturated fatty acids, and decreased saturated fatty acids compared with CO and MA. Both SO and LO increased the proportion of total polyunsaturated fatty acids, total conjugated linolenic acid, and cis-9,trans-11 conjugated linoleic acid. Feeding MA clearly increased the Δ9 desaturation of fatty acids. Our results provide compelling evidence that plant oils supplemented to a grass silage-based diet reduce ruminal CH4 emission and milk saturated fatty acids, and increase the proportion of unsaturated fatty acids and total conjugated linoleic acid while not interfering with digestibility, rumen fermentation, rumen microbial quantities, or milk production.

Downregulation of DmMANF in Glial Cells Results in Neurodegeneration and Affects Sleep and Lifespan in Drosophila melanogaster.

In Drosophila melanogaster, mesencephalic astrocyte-derived neurotrophic factor (DmMANF) is an evolutionarily conserved ortholog of mammalian MANF and cerebral dopamine neurotrophic factor (CDNF), which have been shown to promote the survival of dopaminergic neurons in the brain. We observed especially high levels of DmMANF in the visual system of Drosophila, particularly in the first optic neuropil (lamina). In the lamina, DmMANF was found in glial cells (surface and epithelial glia), photoreceptors and interneurons. Interestingly, silencing of DmMANF in all neurons or specifically in photoreceptors or L2 interneurons had no impact on the structure of the visual system. However, downregulation of DmMANF in glial cells induced degeneration of the lamina. Remarkably, this degeneration in the form of holes and/or tightly packed membranes was observed only in the lamina epithelial glial cells. Those membranes seem to originate from the endoplasmic reticulum, which forms autophagosome membranes. Moreover, capitate projections, the epithelial glia invaginations into photoreceptor terminals that are involved in recycling of the photoreceptor neurotransmitter histamine, were less numerous after DmMANF silencing either in neurons or glial cells. The distribution of the alpha subunit of Na+/K+-ATPase protein in the lamina cell membranes was also changed. At the behavioral level, silencing of DmMANF either in neurons or glial cells affected the daily activity/sleep pattern, and flies showed less activity during the day but higher activity during the night than did controls. In the case of silencing in glia, the lifespan of flies was also shortened. The obtained results showed that DmMANF regulates many functions in the brain, particularly those dependent on glial cells.

In vivo screening reveals interactions between Drosophila Manf and genes involved in the mitochondria and the ubiquinone synthesis pathway.

BACKGROUND: Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. Previous studies indicate that mammalian MANF and CDNF support and protect brain dopaminergic system in non-cell-autonomous manner. However, MANF has also been shown to function intracellularly in the endoplasmic reticulum. To date, the knowledge on the interacting partners of MANF/CDNF and signaling pathways they activate is rudimentary. Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. RESULTS: We first show that DmManf plays a role in the development of Drosophila wing. We exploited this function by using Drosophila UAS-RNAi lines and discovered novel genetic interactions of DmManf with genes known to function in the mitochondria. We also found evidence of an interaction between DmManf and the Drosophila homologue encoding Ku70, the closest structural homologue of SAP domain of mammalian MANF. CONCLUSIONS: In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies.

Dietary forage to concentrate ratio and sunflower oil supplement alter rumen fermentation, ruminal methane emissions, and nutrient utilization in lactating cows.

The effects of supplementing high- or low-concentrate diets with sunflower oil (SO) on rumen fermentation, nutrient utilization, and ruminal methane (CH4) emissions in lactating cows were examined. Four multiparous Nordic Red dairy cows fitted with rumen cannulae were used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments and 35-d periods. Experimental treatments comprised iso-nitrogenous total mixed rations based on grass silage with forage to concentrate ratio of 65:35 or 35:65 supplemented with 0 or 50 g/kg diet DM of SO. Apparent ruminal OM and starch digestibility was greater (P < 0.05) with high- than low-concentrate diets but was unaffected by SO. Inclusion of SO in high-concentrate diet decreased (P ≤ 0.05) apparent total tract OM, fiber, and GE, and apparent ruminal fiber digestibility. High-concentrate diets and SO shifted (P < 0.05) fiber digestion from rumen to the hindgut. High-concentrate diet resulted in a lower rumen pH and elevated total rumen VFA concentration compared with low-concentrate diet, whereas SO increased rumen pH and decreased rumen VFA concentration when included in high-, but not low-concentrate diet (P < 0.05 for interaction). High-concentrate diet reduced rumen ammonia-N (P < 0.01) and molar proportion of acetate to propionate (P < 0.01), and decreased (P < 0.05) ruminal CH4 emissions when expressed as g/d or g/kg OM digested in the rumen. With both low- and high-concentrate diets, SO reduced (P < 0.05) daily emissions of CH4 as g/d or g/kg OM digested in the rumen, but SO reduced CH4 emissions expressed as g/kg OM intake, OM digested in total digestive tract, energy-corrected milk or % of GE intake only with low-concentrate diet (P ≤ 0.05 for interaction). In conclusion, replacing grass silage with concentrates led to a reduction in daily ruminal CH4 emissions that were accompanied by a shift in rumen fermentation toward the synthesis of propionate, and decreases in rumen pH and fiber digestion. Sunflower oil was effective in reducing daily CH4 emissions in lactating cows which was accompanied by a noticeable lower feed intake with high- but not low-concentrate diet. Overall the effects of SO and greater proportion of concentrates in the diet on daily CH4 emissions were additive but the additivity declined or vanished when different indices of CH4 emission intensity were considered. Consequently, SO was more effective in reducing CH4 emissions when low-concentrate diet was fed.

Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster.

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR.

Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor that supports and protects dopaminergic neurons. The Drosophila MANF (DmMANF) null mutant animals die during early development, and DmMANF is required for the maintenance of dopamine positive neurites. The aim of this study was to investigate the role of DmMANF during later developmental stages. Here we report that DmMANF expression in the adult brain is much wider than in the embryonic and larval stages. It is expressed in both glia and neurons including dopaminergic neurons. Clonal analysis showed that DmMANF is not required cell-autonomously for the differentiation of either glia or dopaminergic neurons. In addition, DmMANF overexpression resulted in no apparent abnormal dopaminergic phenotype while DmMANF silencing in glia resulted in prolonged larval stage.

MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain.

Glia are abundant cells in the brain of animals ranging from flies to humans. They perform conserved functions not only in neural development and wiring, but also in brain homeostasis. Here we show that by manipulating gene expression in glia, a previously unidentified cell type appears in the Drosophila brain during metamorphosis. More specifically, this cell type appears in three contexts: (1) after the induction of either immunity, or (2) autophagy, or (3) by silencing of neurotrophic factor DmMANF in glial cells. We call these cells MANF immunoreactive Cells (MiCs). MiCs are migratory based on their shape, appearance in brain areas where no cell bodies exist and the nuclear localization of dSTAT. They are labeled with a unique set of molecular markers including the conserved neurotrophic factor DmMANF and the transcription factor Zfh1. They possess the nuclearly localized protein Relish, which is the hallmark of immune response activation. They also express the conserved engulfment receptor Draper, therefore indicating that they are potentially phagocytic. Surprisingly, they do not express any of the common glial and neuronal markers. In addition, ultrastructural studies show that MiCs are extremely rich in lysosomes. Our findings reveal critical molecular and functional components of an unusual cell type in the Drosophila brain. We suggest that MiCs resemble macrophages/hemocytes and vertebrate microglia based on their appearance in the brain upon genetically challenged conditions and the expression of molecular markers. Interestingly, macrophages/hemocytes or microglia-like cells have not been reported in the fly nervous system before.

Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster.

Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. Here, we report critical roles for lin-28 during oogenesis. We found that let-7 maturation was increased in lin-28 null mutant fly ovaries. We showed that lin-28 null mutant female flies displayed reduced fecundity, due to defects in egg chamber formation. More specifically, we demonstrated that in mutant ovaries, the egg chambers fuse during early oogenesis resulting in abnormal late egg chambers. We also showed that this phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. We suggest a model in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. Our results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation.

Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.

Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo.

Gene expression analysis of Drosophilaa Manf mutants reveals perturbations in membrane traffic and major metabolic changes.

BACKGROUND: MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. RESULTS: The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the endoplasmic reticulum (ER) stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. CONCLUSIONS: We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome the UPR especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.

Characterization of Drosophila GDNF receptor-like and evidence for its evolutionarily conserved interaction with neural cell adhesion molecule (NCAM)/FasII.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-ß superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, we have cloned and characterized the first invertebrate Gfr-like cDNA (DmGfrl) from Drosophila melanogaster and generated a DmGfrl mutant allele. RESULTS: We found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediated neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. We generated a DmGfrl null allele by genomic FLP deletion and found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility. CONCLUSION: Our results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that we describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis. SIGNIFICANCE: These results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling complexes.

Gene expression profiling of U12-type spliceosome mutant Drosophila reveals widespread changes in metabolic pathways.

BACKGROUND: The U12-type spliceosome is responsible for the removal of a subset of introns from eukaryotic mRNAs. U12-type introns are spliced less efficiently than normal U2-type introns, which suggests a rate-limiting role in gene expression. The Drosophila genome contains about 20 U12-type introns, many of them in essential genes, and the U12-type spliceosome has previously been shown to be essential in the fly. METHODOLOGY/PRINCIPAL FINDINGS: We have used a Drosophila line with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, to investigate the impact of U12-type introns on gene expression at the organismal level during fly development. This line exhibits progressive accumulation of unspliced U12-type introns during larval development and the death of larvae at the third instar stage. Surprisingly, microarray and RT-PCR analyses revealed that most genes containing U12-type introns showed only mild perturbations in the splicing of U12-type introns. In contrast, we detected widespread downstream effects on genes that do not contain U12-type introns, with genes related to various metabolic pathways constituting the largest group. CONCLUSIONS/SIGNIFICANCE: U12-type intron-containing genes exhibited variable gene-specific responses to the splicing defect, with some genes showing up- or downregulation, while most did not change significantly. The observed residual U12-type splicing activity could be explained with the mutant U6atac allele having a low level of catalytic activity. Detailed analysis of all genes suggested that a defect in the splicing of the U12-type intron of the mitochondrial prohibitin gene may be the primary cause of the various downstream effects detected in the microarray analysis.

Drosophila twinfilin is required for cell migration and synaptic endocytosis.

Precise actin regulation is essential for diverse cellular processes such as axonal growth, cell migration and endocytosis. twinfilin (twf) encodes a protein that sequesters actin monomers, but its in vivo functions are unclear. In this study, we characterized twf-null mutants in a metazoan for the first time and found that Drosophila twf negatively regulates F-actin formation in subcellular regions of rapid actin turnover in three different systems, namely postsynaptic neuromuscular junction (NMJ) synapses, migratory border cells and epithelial follicle cells. Loss of twf function results in defects in axonal growth in the brain and border cell migration in the ovary. Additionally, we found that the actin-dependent postsynaptic localization of glutamate receptor GluRIIA, but not GluRIIB, was specifically reduced in twf mutants. More importantly, we showed that twf mutations caused significantly reduced presynaptic endocytosis at NMJ synapses, as detected using the fluorescent dye FM1-43 uptake assay. Furthermore, electrophysiological analysis under high-frequency stimulation showed compromised neurotransmission in twf mutant synapses, confirming an insufficient replenishment of synaptic vesicles. Together, our results reveal that twinfilin promotes actin turnover in multiple cellular processes that are highly dependent on actin dynamics.

Evidence that DmMANF is an invertebrate neurotrophic factor supporting dopaminergic neurons.

In vertebrates the development and function of the nervous system is regulated by neurotrophic factors (NTFs). Despite extensive searches no neurotrophic factors have been found in invertebrates. However, cell ablation studies in Drosophila suggest trophic interaction between neurons and glia. Here we report the invertebrate neurotrophic factor in Drosophila, DmMANF, homologous to mammalian MANF and CDNF. DmMANF is expressed in glia and essential for maintenance of dopamine positive neurites and dopamine levels. The abolishment of both maternal and zygotic DmMANF leads to the degeneration of axonal bundles in the embryonic central nervous system and subsequent nonapoptotic cell death. The rescue experiments confirm DmMANF as a functional ortholog of the human MANF gene thus opening the window for comparative studies of this protein family with potential for the treatment of Parkinson's disease.

Molecular anatomy of the cytoplasmic domain of bovine growth hormone receptor, a quantitative trait locus.

Quantitative trait loci (QTL) studies have indicated growth hormone receptor (GHR) as a candidate gene affecting cattle milk yield and composition. In order to characterize genetic variation at GHR in cattle, we studied European and East African breeds with different histories of selection, and Bos grunniens, Ovis aries, Sus scrofa, Bison bison and Rangifer tarandus as references. We sequenced most of the cytoplasmic domain (900 bp of exon 10), 89 bp of exon 8, including the putative causative mutation for the QTL effect, and 390 bp of intron 8 for comparison. In the cytoplasmic domain, seven synonymous and seven non-synonymous single nucleotide polymorphisms (SNP) were identified in cattle. Three non-synonymous SNPs were found in sheep and one synonymous SNP in yak, while other studied species were monomorphic. Three major haplotypes were observed, one unique to African breeds, one unique to European breeds and one shared. Bison and yak haplotypes are derivatives of the European haplotype lineage. Most of the exon 10 non-synonymous cattle SNPs appear at phylogenetically highly conserved sites. The polymorphisms in exon 10 cluster around a ruminant-specific tyrosine residue, suggesting that this site may act as an additional signalling domain of GHR in ruminants. Alternative explanations for the persistent polymorphism include balancing selection, hitch-hiking, pleiotropic or sexually antagonistic fitness effects or relaxed functional constraints.

Prioritization for conservation of northern European cattle breeds based on analysis of microsatellite data.

Northern European indigenous cattle breeds are currently endangered and at a risk of becoming extinct. We analyzed variation at 20 microsatellite loci in 23 indigenous, 3 old imported, and 9 modern commercial cattle breeds that are presently distributed in northern Europe. We measured the breeds' allelic richness and heterozygosity, and studied their genetic relationships with a neighbor-joining tree based on the Chord genetic distance matrix. We used the Weitzman approach and the core set diversity measure of Eding et al. (2002) to quantify the contribution of each breed to the maximum amount of genetic diversity and to identify breeds important for the conservation of genetic diversity. We defined 11 breeds as a "safe set" of breeds (not endangered) and estimated a reduction in genetic diversity if all nonsafe (endangered) breeds were lost. We then calculated the increase in genetic diversity by adding one by one each of the nonsafe breeds to the safe set (the safe-set-plus-one approach). The neighbor-joining tree grouped the northern European cattle breeds into Black-and-White type, Baltic Red, and Nordic cattle groups. Väne cattle, Bohus Poll, and Danish Jersey had the highest relative contribution to the maximum amount of genetic diversity when the diversity was quantified by the Weitzman diversity measure. These breeds not only showed phylogenetic distinctiveness but also low within-population variation. When the Eding et al. method was applied, Eastern Finncattle and Lithuanian White Backed cattle contributed most of the genetic variation. If the loss of the nonsafe set of breeds happens, the reduction in genetic diversity would be substantial (72%) based on the Weitzman approach, but relatively small (1.81%) based on the Eding et al. method. The safe set contained only 66% of the observed microsatellite alleles. The safe-set-plus-one approach indicated that Bohus Poll and Väne cattle contributed most to the Weitzman diversity, whereas the Eastern Finncattle contribution was the highest according to the Eding et al. method. Our results indicate that both methods of Weitzman and Eding et al. recognize the importance of local populations as a valuable resource of genetic variation.

The Cdi/TESK1 kinase is required for Sevenless signaling and epithelial organization in the Drosophila eye.

How cellular behaviors such as cell-to-cell communication, epithelial organization and cell shape reorganization are coordinated during development is poorly understood. The developing Drosophila eye offers an ideal model system to study these processes. Localized actin polymerization is required to constrict the apical surface of epithelial cells of the eye imaginal disc to maintain the refined arrangement of the developing ommatidia. The identity of each photoreceptor cell within the epithelium is determined by cell-to-cell contacts involving signal transduction events. The R7 photoreceptor cell requires the activity of the Sevenless RTK to adopt a proper cell fate. We performed an EP screen for negative regulators of this inductive process, and we identified the serine/threonine kinase Center divider (cdi) as a suppressor of the phenotype caused by an activated Sevenless receptor. Cdi is homologous to the human testis-specific kinase 1 (TESK1), a member of the LIM kinases involved in cytoskeleton control through ADF/cofilin phosphorylation. We have analyzed the effects of gain- and loss-of-function of cdi and found alterations in actin organization and in the adherens junctions proteins DE-cadherin and beta-catenin, as well as in Sevenless apical localization. Interference with the function of the ADF/cofilin phosphatase Slingshot (ssh), which antagonizes Cdi, also results in a suppression of signaling triggered by the Sevenless RTK. Our results reveal a critical interplay between the localization of molecules involved in epithelial organization and signal transduction.