Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells.

The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with 3H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the "lipogenic" enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.

Malic enzyme and fatty acid synthase in the uropygial gland and liver of embryonic and neonatal ducklings. Tissue-specific regulation of gene expression.

Malic enzyme [L-malate-NADP oxidoreductase (decarboxylating), EC 1.1.1.40] and fatty acid synthase activities were barely detectable in the uropygial gland of duck embryos until 4 or 5 days before hatching, when they began to increase. These activities increased about 30- and 140-fold, respectively, by the day of hatching. Malic enzyme and fatty acid synthase activities were also very low in embryonic liver. However, hepatic malic enzyme activity did not increase until the newly hatched ducklings were fed. Hepatic fatty acid synthase began to increase the day before hatching and the rate of increase in enzyme activity accelerated markedly when the newly hatched ducklings were fed. Starvation of newly hatched or 12-day-old ducklings had no effect on the activities of malic enzyme and fatty acid synthase in the uropygial gland but markedly inhibited these activities in liver. Changes in the concentrations of both enzymes and in the relative synthesis rates of fatty acid synthase correlated with enzyme activities in both uropygial gland and liver. Developmental patterns for sequence abundance of malic enzyme and fatty acid synthase mRNAs in uropygial gland and liver were similar to those for their respective enzyme activities. Starvation of 4-day-old ducklings had no significant effect on the abundance of these mRNAs in uropygial gland but caused a pronounced decrease in their abundance in liver. It is concluded that developmental and nutritional regulation of these enzymes is tissue specific and occurs primarily at a pretranslational level in both uropygial gland and liver.

Molecular cloning of cDNA sequences for avian malic enzyme. Nutritional and hormonal regulation of malic enzyme mRNA levels in avian liver cells in vivo and in culture.

A double-stranded cDNA library constructed from the total poly(A+) RNA of goose uropygial gland was screened for recombinants containing sequences complementary to malic enzyme mRNA. Replicate arrays of 1400 colonies were hybridized independently with 32P-labeled cDNAs copied from two populations of hepatic RNA derived from tissues which differed by about 35-fold with respect to the relative synthesis of malic enzyme. Forty-eight of the colonies which gave differential signals were further screened by hybrid-selected translation. DNA from one of these contained an insert of 970 base pairs and selected an mRNA which directed the synthesis of malic enzyme in a cell-free system. The malic enzyme sequences were subcloned into the single-stranded bacteriophage M13mp8. The subclones were used to prepare 32P-labeled single-stranded hybridization probe. Northern analysis indicated that malic enzyme mRNA from both goose and chicken is about 2100 bases in length. Hepatic malic enzyme mRNA concentration is stimulated 30- to 50-fold or more when neonatal chicks or goslings, respectively, are fed for 24 h. When added to chick embryo hepatocytes in culture, triiodothyronine stimulated malic enzyme mRNA accumulation by more than 100-fold. Glucagon inhibited the thyroid hormone-stimulated accumulation of malic enzyme mRNA by 99%. In all instances, malic enzyme mRNA concentration was closely correlated with the relative rate of malic enzyme synthesis. These results suggest that nutritional and hormonal regulation of malic enzyme synthesis occurs at the pretranslational level.

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration.

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.