Hepatic Gene Expression Profiling of Riboflavin-deficient and Riboflavin-rescued Chicken Embryos at 9, 11, 13 and 15 days of incubation. Gallus gallus

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. Overall design: A reference RNA design was used for microarray hybridizations, where the same reference RNA pool was co-hybridized to each target samples on an microarray. RNA pool was made from an equal amount of high-quality amplified RNA (aRNA), derived from all samples. Reference RNA pool was labeled with Alexa 647 while each of target samples was labeled with Alexa 555.