Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering.
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ABSTRACT: A strain of Leghorn chickens (rd/rd), unable to produce a functional riboflavin-binding protein, lays riboflavin-deficient eggs, in which all embryos suddenly die at mid-incubation (days 13-15). This malady, caused by riboflavin deficiency, leads to excessive lipid accumulation in liver, impaired ?-oxidation of lipid, and severe hypoglycemia prior to death. We have used high-density chicken microarrays for time-course transcriptional scans of liver in chicken embryos between days 9-15 during this riboflavin-deficiency-induced metabolic catastrophe. For comparison, half of rd/rd embryos (n?=?16) were rescued from this calamity by injection of riboflavin just prior to incubation of fertile eggs from rd/rd hens.No significant differences were found between hepatic transcriptomes of riboflavin-deficient and riboflavin-rescued embryos at the first two ages (days 9 and 11). Overall, we found a 3.2-fold increase in the number of differentially expressed hepatic genes between day 13 (231 genes) and day 15 (734 genes). Higher expression of genes encoding the chicken flavoproteome was more evident in rescued- (15 genes) than in deficient-embryos (4 genes) at day 15. Diminished activity of flavin-dependent enzymes in riboflavin-deficient embryos blocks catabolism of yolk lipids, which normally serves as the predominant source of energy required for embryonic development.Riboflavin deficiency in mid-stage embryos leads to reduced expression of numerous genes controlling critical functions, including ?-oxidation of lipids, blood coagulation and feathering. Surprisingly, reduced expression of feather keratin 1 was found in liver of riboflavin-deficient embryos at e15, which could be related to their delayed feathering and sparse clubbed down. A large number of genes are expressed at higher levels in liver of riboflavin-deficient embryos; these up-regulated genes control lipid storage/transport, gluconeogenesis, ketogenesis, protein catabolism/ubiquitination and cell death.
Transcriptional profiling of liver in riboflavin-deficient chicken embryos explains impaired lipid utilization, energy depletion, massive hemorrhaging, and delayed feathering.
<h4>Background</h4>A strain of Leghorn chickens (rd/rd), unable to produce a functional riboflavin-binding protein, lays riboflavin-deficient eggs, in which all embryos suddenly die at mid-incubation (days 13-15). This malady, caused by riboflavin deficiency, leads to excessive lipid accumulation in liver, impaired β-oxidation of lipid, and severe hypoglycemia prior to death. We have used high-density chicken microarrays for time-course transcriptional scans of liver in chicken embryos between d ...[more]
Project description:This study aimed to identify key genes and pathways related to essential functions of riboflavin during the development of chicken embryos. We used as a unique model a strain of mutant chickens (rd/rd) which is unable to produce a riboflavin-binding protein and lays riboflavin-deficient eggs in which the embryos suddenly die between 13 and 15 days of incubation (e13 and e15, respectively). In these embryos, the diminished activity of flavin-dependent enzymes, particularly those associated with β-oxidation of fatty acid, limits the catabolism of yolk lipids that normally serve as the predominant source of energy required for growth. This malady leads to excessive lipid accumulation in liver and severe hypoglycemia prior to death. This lethal effect can be rescued by an injection of riboflavin just prior to incubation of fertile rd/rd eggs. Microarray-based hepatic transcriptome profiling of riboflavin-deficient (Rf-) and riboflavin-rescue (Rf+) chicken embryos were carried out at 9, 11, 13 and 15 days of incubation. Our microarray analysis identified a dramatic change in gene expression between the Rf- and Rf+ groups on e13 and e15 with 221 and 929 differentially expressed genes (DEGs), respectively. PPAR-gamma, apolipoprotein AV, adipophilin and sterol 14-alpha demethylase were up-regulated, while alpha2 antiplasmin, alpha1 antitrypsin, alpha2 microglobulin, TIP120 and GHRG1 were down-regulated in the Rf- embryos which show an increased lipid accumulation in liver that may be resulted from the disruption in fatty acid beta-oxidation. Genes involved in detoxification, the immune response and blood coagulation were down-regulated, whereas several phosphatase genes were increased. There were no apparent systematic changes in expression of genes associated with the flavin-dependent enzymes, with an exception of an increase in medium chain acyl-CoA dehydrogenase.
Project description:Background:Henny feathering in chickens is determined by a dominant mutation that transforms male-specific plumage to female-like plumage. Previous studies indicated that this phenotype is caused by ectopic expression in skin of CYP19A1 encoding aromatase that converts androgens to estrogen and thereby inhibits the development of male-specific plumage. A long terminal repeat (LTR) from an uncharacterized endogenous retrovirus (ERV) insertion was found in an isoform of the CYP19A1 transcript from henny feathering chicken. However, the complete sequence and the genomic position of the insertion were not determined. Results:We used publicly available whole genome sequence data to determine the flanking sequences of the ERV, and then PCR amplified the entire insertion and sequenced it using Nanopore long reads and Sanger sequencing. The 7524?bp insertion contains an intact endogenous retrovirus that was not found in chickens representing 31 different breeds not showing henny feathering or in samples of the ancestral red junglefowl. The sequence shows over 99% sequence identity to the avian leukosis virus ev-1 and ev-21 strains, suggesting a recent integration. The ERV 3'LTR, containing a powerful transcriptional enhancer and core promoter with TATA box together with binding sites for EFIII and Ig/EBP inside the CYP19A1 5' untranslated region, was detected partially in an aromatase transcript, which present a plausible explanation for ectopic expression of aromatase in non-ovarian tissues underlying the henny feathering phenotype. Conclusions:We demonstrate that the henny feathering allele harbors an insertion of an intact avian leukosis virus at the 5'end of CYP19A1. The presence of this ERV showed complete concordance with the henny feathering phenotype both within a pedigree segregating for this phenotype and across breeds.
Project description:Sex-linked slow (SF) and fast (FF) feathering rates at hatch have been widely used in poultry breeding for autosexing at hatch. In chicken, the sex-linked K (SF) and k+ (FF) alleles are responsible for the feathering rate phenotype. Allele K is dominant and a partial duplication of the prolactin receptor gene has been identified as the causal mutation. Interestingly, some domesticated turkey lines exhibit similar slow- and fast-feathering phenotypes, but the underlying genetic components and causal mutation have never been investigated. In this study, our aim was to investigate the molecular basis of feathering rate at hatch in domestic turkey.We performed a sequence-based case-control association study and detected a genomic region on chromosome Z, which is statistically associated with rate of feathering at hatch in turkey. We identified a 5-bp frameshift deletion in the prolactin receptor (PRLR) gene that is responsible for slow feathering at hatch. All female cases (SF turkeys) were hemizygous for this deletion, while 188 controls (FF turkeys) were hemizygous or homozygous for the reference allele. This frameshift mutation introduces a premature stop codon and six novel amino acids (AA), which results in a truncated PRLR protein that lacks 98 C-terminal AA.We present the causal mutation for feathering rate in turkey that causes a partial C-terminal loss of the prolactin receptor, and this truncated PRLR protein is strikingly similar to the protein encoded by the slow feathering K allele in chicken.
Project description:The concentration of riboflavin and riboflavin-binding protein were determined in the plasma, egg yolk and albumen from hens fed a riboflavin-deficient diet (1.2 mg/kg) supplemented with 0, 1, 2, 3, 10 and 40 mg of riboflavin/kg. We observed that the deposition of riboflavin in egg yolk and albumen is dependent on dietary riboflavin and reaches half-maximal values at about 2 mg of supplemental riboflavin/kg. The maximal amount of riboflavin deposited in the yolk is limited stoichiometrically by the amount of riboflavin-binding protein, whereas the maximum amount of riboflavin deposited in albumen is limited by other factors before saturation occurs. The amount of riboflavin-binding protein in yolk and albumen is independent of dietary riboflavin. If there is a specific oocyte receptor for riboflavin-binding protein, it cannot distinguish between the apo and holo forms of the protein. Riboflavin-binding protein is about six times more concentrated in yolk than in plasma.
Project description:Sex-linked phenotypes of late feathering (LF) and early feathering (EF) are controlled by a pair of alleles K and k+. Autosexing based on the feathering rate is widely used in poultry production. It is reported that a tandem duplication of 176,324 base pairs linked to the K locus is responsible for LF expression and could be used as a molecular marker to detect LF chicken. So far, there is no genotyping method that can accurately and stably identify the LF homozygote and heterozygote in all chicken breeds. In the present study, a multiplex PCR test was developed to identify EF, LF homozygote, and heterozygote according to electrophoretic bands and the relative height of the peaks by Sanger sequencing. We tested 413 chickens of six native Chinese breeds with this method. The identification was consistent with the sex and phenotype records of the chickens. Band density analysis was performed, and the results supported our genotyping using the new assay. In order to further verify the accuracy of this test in distinguishing homozygote and heterozygote males, 152 LF males were mated with EF females, and the results of the offspring's phenotypes were consistent with our expectations. Our results support tandem duplication as molecular markers of LF, and this new test is applicable to all LF chickens associated with tandem duplication.
Project description:BACKGROUND:Early feathering and late feathering in chickens are sex-linked phenotypes, which have commercial application in the poultry industry for sexing chicks at hatch and have important impacts on performance traits. However, the genetic mechanism controlling feather development and feathering patterns is unclear. Here, miRNA and mRNA expression profiles in chicken wing skin tissues were analysed through high-throughput transcriptomic sequencing, aiming to understand the biological process of follicle development and the formation of different feathering phenotypes. RESULTS:Compared to the N1 group with no primary feathers extending out, 2893 genes and 31 miRNAs displayed significantly different expression in the F1 group with primary feathers longer than primary-covert feathers, and 1802 genes and 11 miRNAs in the L2 group displayed primary feathers shorter than primary-covert feathers. Only 201 altered genes and 3 altered miRNAs were identified between the N1 and L2 groups (fold change > 2, q value < 0.01). Both sequencing and qPCR tests revealed that PRLR was significantly decreased in the F1 and L2 groups compared to the N1 group, whereas SPEF2 was significantly decreased in the F1 group compared to the N1 or L2 group. Functional analysis revealed that the altered genes or targets of altered miRNAs were involved in multiple biological processes and pathways related to feather growth and development, such as the Wnt signalling pathway, the TGF-beta signalling pathway, the MAPK signalling pathway, epithelial cell differentiation, and limb development. Integrated analysis of miRNA and mRNA showed that 14 pairs of miRNA-mRNA negatively interacted in the process of feather formation. CONCLUSIONS:Transcriptomic sequencing of wing skin tissues revealed large changes in F1 vs. N1 and L2 vs. N1, but few changes in F1 vs. L2 for both miRNA and mRNA expression. PRLR might only contribute to follicle development, while SPEF2 was highly related to the growth rate of primary feathers or primary-covert feathers and could be responsible for early and late feather formation. Interactions between miR-1574-5p/NR2F, miR-365-5p/JAK3 and miR-365-5p/CDK6 played important roles in hair or feather formation. In all, our results provide novel evidence to understand the molecular regulation of follicle development and feathering phenotype.
Project description:Despite insistent warnings from climate scientists, the global environmental situation is further deteriorating. To date, only very few studies have investigated the impact of warnings on sustainable decision-making in controlled laboratory settings. Moreover, the few existing studies mainly looked at average warning reactions rather than taking individual differences into account. Here, we investigated individual differences in the reaction to resource depletion warnings and scrutinized the impact of emotions on behavioural changes by applying a resource dilemma task with warnings. Data-driven and model-free cluster analyses identified four different types of consumption behaviour. Importantly, guilt was positively related to sustainable decision-making after warnings. In contrast, a lack of guilt was associated with no behavioural change or even worse with more unsustainable behaviour after warnings. These findings contribute to the debate over effective climate change communication by demonstrating that issuing warnings about the climate crisis only leads to the intended behavioural changes if people experience guilt.
Project description:We introduce a modeling approach for characterizing heterogeneity in healthcare utilization using massive medical claims data. We first translate the medical claims observed for a large study population and across five years into individual-level discrete events of care called utilization sequences. We model the utilization sequences using an exponential proportional hazards mixture model to capture heterogeneous behaviors in patients' healthcare utilization. The objective is to cluster patients according to their longitudinal utilization behaviors and to determine the main drivers of variation in healthcare utilization while controlling for the demographic, geographic, and health characteristics of the patients. Due to the computational infeasibility of fitting a parametric proportional hazards model for high-dimensional, large sample size data we use an iterative one-step procedure to estimate the model parameters and impute the cluster membership. The approach is used to draw inferences on utilization behaviors of children in the Medicaid system with persistent asthma across six states. We conclude with policy implications for targeted interventions to improve adherence to recommended care practices for pediatric asthma.
Project description:Key roles for fibronectin and its integrin receptors have been postulated in the multiple cell-matrix interactions essential for chick embryo morphogenesis. However, mechanistic studies of these processes have been hampered by the current absence of sequence data and chicken cDNA clones for the major fibronectin receptor subunit, integrin ?5 (ITGA5). We report here the sequence, endogenous expression pattern, and transfection of full-length chicken integrin ?5. During early chicken embryonic development, ?5 is highly expressed in cranial neural folds and migrating neural crest cells, suggesting potential roles in neural crest formation and migration. In fact, over-expression of this integrin in early neural tube selectively induces BMP5, a growth factor recently implicated in neural crest formation. Availability of these ?5 integrin tools should facilitate studies of its functions in early embryonic development.
